Orthogonal translation components for the in vivo incorporation of unnatural amino acids

ABSTRACT

The invention relates to orthogonal pairs of tRNAs and aminoacyl-tRNA synthetase that can incorporate unnatural amino acid into proteins produced in eubacterial host cells such as  E. coli , or in a eukaryotic host such as a yeast cell. The invention provides, for example but not limited to, novel orthogonal synthetases, methods for identifying and making the novel synthetases, methods for producing proteins containing unnatural amino acids, and translation systems.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Divisional application from, and claims benefitand priority from, U.S. application 11/665,083, Orthogonal TranslationComponents for the In Vivo Incorporation of Unnatural Amino Acids, toPeter Schultz, et al., filed Jun. 18, 2007now U.S. Pat. No. 8,216,804,which was a section 371 application from PCT/US05/39210, filed Oct. 27,2005, and which claims priority to and benefit of United StatesProvisional Patent Application Serial No. 60/622,738, filed Oct. 27,2004, the disclosure of which is incorporated herein by reference in itsentirety for all purposes.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

This invention was made with government support under Grant No. DE-FG03-00ER45601 awarded by the Department of Energy, and under Grant Nos.GM062159 and AI066507 awarded by the National Institutes of Health. Thegovernment has certain rights in this invention.

FIELD OF THE INVENTION

The invention is in the field of translation biochemistry. The inventionrelates to compositions and methods for making and using orthogonaltRNAs, orthogonal aminoacyl-tRNA synthetases, and pairs thereof, thatincorporate unnatural amino acids into proteins. The invention alsorelates to methods of producing proteins in cells using such pairs andproteins made by the methods.

BACKGROUND OF THE INVENTION

The study of protein structure and function has historically relied uponthe properties and reaction chemistries that are available using thereactive groups of the naturally occurring amino acids. Unfortunately,every known organism, from bacteria to humans, encodes the same twentycommon amino acids (with the rare exceptions of selenocysteine (see,e.g., A. Bock et al., (1991), Molecular Microbiology 5:515-20) andpyrrolysine (see, e.g., G. Srinivasan, et al., (2002), Science296:1459-62). This limited selection of R-groups has restricted thestudy of protein structure and function, where the studies are confinedby the chemical properties of the naturally occurring amino acids, e.g.,the natural amino acids limit the ability to make highly targetedprotein modifications to the exclusion of all other amino acids in aprotein. Additionally, the natural amino acids are limited in theirfunctional activities, e.g., fluorescence, metal chelating,redox-potential, photocaging, etc.

Most modification reactions currently used in the art for the selectivemodification of proteins involve covalent bond formation betweennucleophilic and electrophilic reaction partners that target naturallyoccurring nucleophilic residues in the protein amino acid side chains,e.g., the reaction of α-halo ketones with histidine or cysteine sidechains. Selectivity in these cases is determined by the number andaccessibility of the nucleophilic residues in the protein.Unfortunately, naturally occurring proteins frequently contain poorlypositioned (e.g., inaccessible) reaction sites or multiple reactiontargets (e.g., lysine, histidine and cysteine residues), resulting inpoor selectivity in the modification reactions, making highly targetedprotein modification by nucleophilic/electrophilic reagents difficult.Furthermore, the sites of modification are typically limited to thenaturally occurring nucleophilic side chains of lysine, histidine orcysteine. Modification at other sites is difficult or impossible.

What is needed in the art are new strategies for incorporation ofunnatural amino acids into proteins for the purpose of modifying andstudying protein structure and function, where the unnatural amino acidshave novel properties, e.g., biological properties, not found in thenaturally occurring amino acids. There is a considerable need in the artfor the creation of new strategies for protein modification reactionsthat modify proteins in a highly selective fashion, and furthermore,modify proteins under physiological conditions. What is needed in theart are novel methods for producing protein modifications, where themodifications are highly specific, e.g., modifications where none of thenaturally occurring amino acids are subject to cross reactions or sidereactions. Novel chemistries for highly specific protein modificationstrategies can find a wide variety of applications in the study ofprotein structure and function.

One strategy to overcome these limitations is to expand the genetic codeand add amino acids that have distinguishing physical, chemical orbiological properties to the biological repertoire. This approach hasproven feasible using orthogonal tRNA's and corresponding novelorthogonal aminoacyl-tRNA synthetases to add unnatural amino acids toproteins using the in vivo protein biosynthetic machinery of a hostcell, e.g., the eubacteria Escherichia coli (E. coli), yeast ormammalian cells. This approach is described in various sources, forexample, Wang et al., (2001), Science 292:498-500; Chin et al., (2002)Journal of the American Chemical Society 124:9026-9027; Chin andSchultz, (2002), ChemBioChem 11:1135-1137; Chin, et al., (2002), PNASUnited States of America 99:11020-11024; and Wang and Schultz, (2002),Chem. Comm., 1-10. See also, International Publications WO 2002/086075,entitled “METHODS AND COMPOSITIONS FOR THE PRODUCTION OF ORTHOGONAL tRNAAMINOACYL-tRNA SYNTHETASE PAIRS;” WO 2002/085923, entitled “IN VIVOINCORPORATION OF UNNATURAL AMINO ACIDS;” WO 2004/094593, entitled“EXPANDING THE EUKARYOTIC GENETIC CODE;” WO 2005/019415, filed Jul. 7,2004; WO 2005/007870, filed Jul. 7, 2004; and WO 2005/007624, filed Jul.7, 2004.

There is a need in the art for the development of orthogonal translationcomponents that incorporate unnatural amino acids into proteins, wherethe unnatural amino acids can be incorporated at a defined position, andwhere the unnatural amino acids impart novel biological properties tothe proteins in which they are incorporated. There is also a need todevelop orthogonal translation components that incorporate unnaturalamino acids with novel chemical properties that allow the amino acid toserve as a target for specific modification to the exclusion of crossreactions or side reactions with other sites in the proteins. There isalso a particular need for protein expression systems that have theability to produce proteins containing unnatural amino acids insignificant quantities that permit their use in therapeutic applicationsand biomedical research. The invention described herein fulfills theseand other needs, as will be apparent upon review of the followingdisclosure.

SUMMARY OF THE INVENTION

The invention provides compositions and methods for incorporatingunnatural amino acids into a growing polypeptide chain in response to aselector codon, e.g., an amber stop codon, in vivo (e.g., in a hostcell). These compositions include pairs of orthogonal-tRNAs (O-tRNAs)and orthogonal aminoacyl-tRNA synthetases (O-RSs) that do not interactwith the host cell translation machinery. That is to say, the O-tRNA isnot charged (or not charged to a significant level) with an amino acid(natural or unnatural) by an endogenous host cell aminoacyl-tRNAsynthetase. Similarly, the O-RSs provided by the invention do not chargeany endogenous tRNA with an amino acid (natural or unnatural) to asignificant or in some cases detectable level. These novel compositionspermit the production of large quantities of proteins havingtranslationally incorporated unnatural amino acids. Depending on thechemical properties of the unnatural amino acid that is incorporated,these proteins find a wide variety of uses, including, for example, astherapeutics and in biomedical research.

In some aspects, the invention provides translation systems. Thesesystems comprise a first orthogonal aminoacyl-tRNA synthetase (O-RS), afirst orthogonal tRNA (O-tRNA), and an unnatural amino acid, where thefirst O-RS preferentially aminoacylates the first O-tRNA with the firstunnatural amino acid. The first unnatural amino acid can be selectedfrom p-ethylthiocarbonyl-L-phenylalanine,p-(3-oxobutanoyl)-L-phenylalanine, 1,5-dansyl-alanine,7-amino-coumarin-alanine, 7-hydroxy-coumarin-alanine,o-nitrobenzyl-serine, O-(2-nitrobenzyl)-L-tyrosine,p-carboxymethyl-L-phenylalanine, m-cyano-L-phenylalanine,p-cyano-L-phenylalanine, biphenyl alanine, 3-amino-L-tyrosine,bipyridylalanine, p-(2-amino-1-hydroxyethyl)-L-phenylalanine andp-isopropylthiocarbonyl-L-phenylalanine.

The translation systems can use components derived from a variety ofsources. In one embodiment, the first O-RS is derived from aMethanococcus jannaschii aminoacyl-tRNA synthetase, e.g., a wild-typeMethanococcus jannaschii tyrosyl-tRNA synthetase. In other embodiments,the O-RS is derived from an E. coli aminoacyl-tRNA synthetase, e.g., awild-type E. coli leucyl-tRNA synthetase. The O-RS used in the systemcan comprise an amino acid sequence selected from SEQ ID NOs: 12, 14,16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 50,52-55, 57, 59-63, and conservative variants of those sequences. In someembodiments, the O-tRNA is an amber suppressor tRNA. In someembodiments, the O-tRNA comprises or is encoded by SEQ ID NO: 1 or 2.

In some aspects, the translation system further comprises a nucleic acidencoding a protein of interest, where the nucleic acid has at least oneselector codon that is recognized by the O-tRNA.

In some aspects, the translation system incorporates a second orthogonalpair (that is, a second O-RS and a second O-tRNA) that utilizes a secondunnatural amino acid, so that the system is now able to incorporate atleast two different unnatural amino acids at different selected sites ina polypeptide. In this dual system, the second O-RS preferentiallyaminoacylates the second O-tRNA with a second unnatural amino acid thatis different from the first unnatural amino acid, and the second O-tRNArecognizes a selector codon that is different from the selector codonrecognized by the first O-tRNA.

In some embodiments, the translation system resides in a host cell (andincludes the host cell). The host cell used in not particularly limited,as long as the O-RS and O-tRNA retain their orthogonality in their hostcell environment. The host cell can be a eubacterial cell, such as E.coli, or a yeast cell, such as Saccharomyces cerevisiae. The host cellcan comprise one or more polynucleotides that encode components of thetranslation system, including the O-RS or O-tRNA. In some embodiments,the polynucleotide encoding the O-RS comprises a nucleotide sequence ofSEQ ID NO: 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41,43, 45, 47, 51, 56 or 58.

The invention also provides methods for producing proteins having one ormore unnatural amino acids at selected positions. These methods utilizethe translation systems described above. Generally, these methods startwith the step of providing a translation system comprising: (i) a firstunnatural amino acid selected from p-ethylthiocarbonyl-L-phenylalanine,p-(3-oxobutanoyl)-L-phenylalanine, 1,5-dansyl-alanine,7-amino-coumarin-alanine, 7-hydroxy-coumarin-alanine,o-nitrobenzyl-serine, O-(2-nitrobenzyl)-L-tyrosine,p-carboxymethyl-L-phenylalanine, m-cyano-L-phenylalanine,p-cyano-L-phenylalanine, biphenylalanine, 3-amino-L-tyrosine,bipyridyalanine, p-(2-amino-1-hydroxyethyl)-L-phenylalanine andp-isopropylthiocarbonyl-L-phenylalanine; (ii) a first orthogonalaminoacyl-tRNA synthetase (O-RS); (iii) a first orthogonal tRNA(O-tRNA), wherein the O-RS preferentially aminoacylates the O-tRNA withthe unnatural amino acid; and, (iv) a nucleic acid encoding the protein,where the nucleic acid comprises at least one selector codon that isrecognized by the first O-tRNA. The method then incorporates theunnatural amino acid at the selected position in the protein duringtranslation of the protein in response to the selector codon, therebyproducing the protein comprising the unnatural amino acid at theselected position.

This methods can be widely applied using a variety of reagents andsteps. In some embodiments, a polynucleotide encoding the O-RS isprovided. In some embodiments, an O-RS derived from a Methanococcusjannaschii aminoacyl-tRNA synthetase is provided, for example, awild-type Methanococcus jannaschii tyrosyl-tRNA synthetase can beprovided. In other embodiments, an O-RS derived from an E. coliaminoacyl-tRNA synthetase is provided, e.g., an O-RS derived from awild-type E. coli leucyl-tRNA synthetase can be provided. In someembodiments, the providing step includes providing an O-RS comprising anamino acid sequence selected from SEQ ID NOs: 12, 14, 16, 18, 20, 22,24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 50, 52-55, 57, 59-63,and conservative variants thereof.

In some embodiments of these methods, the providing a translation systemstep comprises mutating an amino acid binding pocket of a wild-typeaminoacyl-tRNA synthetase by site-directed mutagenesis, and selecting aresulting O-RS that preferentially aminoacylates the O-tRNA with theunnatural amino acid. The selecting step can comprises positivelyselecting and negatively selecting for the O-RS from a pool of resultingaminoacyl-tRNA synthetase molecules following site-directed mutagenesis.In some embodiments, the providing step furnishes a polynucleotideencoding the O-tRNA, e.g., an O-tRNA that is an amber suppressor tRNA,or an O-tRNA that comprises or is encoded by a polynucleotide of SEQ IDNO: 1 or 2. In these methods, the providing step can also furnish anucleic acid comprising an amber selector codon that is utilized by thetranslation system.

These methods can also be modified to incorporate more than oneunnatural amino acid into a protein. In those methods, a secondorthogonal translation system is employed in conjunction with the firsttranslation system, where the second system has different amino acid andselector codon specificities. For example, the providing step caninclude providing a second O-RS and a second O-tRNA, where the secondO-RS preferentially aminoacylates the second O-tRNA with a secondunnatural amino acid that is different from the first unnatural aminoacid, and where the second O-tRNA recognizes a selector codon in thenucleic acid that is different from the selector codon recognized by thefirst O-tRNA.

The methods for producing a protein with an unnatural amino acid canalso be conducted in the context of a host cell. In these cases, a hostcell is provided, where the host cell comprises the unnatural aminoacid, the O-RS, the O-tRNA and the nucleic acid, and where culturing thehost cell results in incorporating the unnatural amino acid. In someembodiments, the providing step comprises providing a eubacterial hostcell (e.g., E. coli) or a yeast host cell. In some embodiments, theproviding step includes providing a host cell that contains apolynucleotide encoding the O-RS. Fore example, the polynucleotideencoding the O-RS can comprise a nucleotide sequence of SEQ ID NOs: 13,15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 51,56 or 58. In some embodiments, the step of providing a translationsystem is accomplished by providing a cell extract.

In some aspects, the invention provides translation systems, where thesystems are for the incorporation of 3-nitro-L-tyrosine orp-nitro-L-phenylalanine. These systems comprise a first orthogonalaminoacyl-tRNA synthetase (O-RS), a first orthogonal tRNA (O-tRNA), andthe unnatural amino acid, where the first O-RS preferentiallyaminoacylates the first O-tRNA with the first unnatural amino acid withan efficiency that is at least 50% of the efficiency observed for atranslation system comprising that same unnatural amino acid, the O-tRNAand an O-RS comprising an amino acid sequence selected from SEQ ID NOs:7-10.

The translation system can use components derived from a variety ofsources. In some embodiments, the first O-RS is derived from aMethanococcus jannaschii aminoacyl-tRNA synthetase, e.g., a wild-typeMethanococcus jannaschii tyrosyl-tRNA synthetase. The O-RS used in thesystem can comprise an amino acid sequence selected from SEQ ID NOs:7-10, and conservative variants of those sequences. In some embodiments,the O-tRNA is an amber suppressor tRNA. In some embodiments, the O-tRNAcomprises or is encoded by SEQ ID NO: 1.

In some aspects, the translation system further comprises a nucleic acidencoding a protein of interest, where the nucleic acid has at least oneselector codon that is recognized by the O-tRNA.

In some aspects, the translation system incorporates a second orthogonalpair (that is, a second O-RS and a second O-tRNA) that utilizes a secondunnatural amino acid, so that the system is now able to incorporate atleast two different unnatural amino acids at different selected sites ina polypeptide. In this dual system, the second O-RS preferentiallyaminoacylates the second O-tRNA with a second unnatural amino acid thatis different from the first unnatural amino acid, and the second O-tRNArecognizes a selector codon that is different from the selector codonrecognized by the first O-tRNA.

In some embodiments, the translation system resides in a host cell (andincludes the host cell). The host cell used is not particularly limited,as long as the O-RS and O-tRNA retain their orthogonality in their hostcell environment. The host cell can be a eubacterial cell, such as E.coli. The host cell can comprise one or more polynucleotides that encodecomponents of the translation system, including the O-RS or O-tRNA. Insome embodiments, the polynucleotide encoding the O-RS comprises anucleotide sequence of SEQ ID NO: 11.

The invention also provides methods for producing proteins having one ormore unnatural amino acids at selected positions. These methods utilizethe translation systems described above. Generally, these methods startwith the step of providing a host cell comprising a translation systemcomprising: (i) a first unnatural amino acid that is 3-nitro-L-tyrosineor p-nitro-L-phenylalanine; (ii) a first orthogonal aminoacyl-tRNAsynthetase (O-RS); (iii) a first orthogonal tRNA (O-tRNA), where theO-RS preferentially aminoacylates the O-tRNA with the unnatural aminoacid with an efficiency that is at least 50% of the efficiency observedfor the host cell comprising the unnatural amino acid, the O-tRNA and anO-RS comprising an amino acid sequence selected from SEQ ID NOs: 7-10;and, (iv) a nucleic acid encoding the protein, where the nucleic acidcomprises at least one selector codon that is recognized by the O-tRNA.The host cell is then grown, and the unnatural amino acid isincorporated at the selected position in the protein during translationof the protein in response to the selector codon, where the selectedposition in the protein corresponds to the position of the selectorcodon in the nucleic acid, thereby producing the protein comprising theunnatural amino acid at the selected position.

These methods can be widely applied using a variety of reagents andsteps. In some embodiments, a polynucleotide encoding the O-RS isprovided. In some embodiments, an O-RS derived from a Methanococcusjannaschii aminoacyl-tRNA synthetase is provided, for example, awild-type Methanococcus jannaschii tyrosyl-tRNA synthetase can beprovided. In some embodiments, the providing step includes providing anO-RS comprising an amino acid sequence selected from SEQ ID NOs: 7-10,and conservative variants thereof.

In some embodiments of these methods, the providing a translation systemstep comprises mutating an amino acid binding pocket of a wild-typeaminoacyl-tRNA synthetase by site-directed mutagenesis, and selecting aresulting O-RS that preferentially aminoacylates the O-tRNA with theunnatural amino acid. The selecting step can comprises positivelyselecting and negatively selecting for the O-RS from a pool of resultingaminoacyl-tRNA synthetase molecules following site-directed mutagenesis.In some embodiments, the providing step furnishes a polynucleotideencoding the O-tRNA, e.g., an O-tRNA that is an amber suppressor tRNA,or an O-tRNA that comprises or is encoded by a polynucleotide of SEQ IDNO: 1. In these methods, the providing step can also furnish a nucleicacid comprising an amber selector codon that is utilized by thetranslation system.

These methods can also be modified to incorporate more than oneunnatural amino acid into a protein. In those methods, a secondorthogonal translation system is employed in conjunction with the firsttranslation system, where the second system has different amino acid andselector codon specificities. For example, the providing step caninclude providing a second O-RS and a second O-tRNA, where the secondO-RS preferentially aminoacylates the second O-tRNA with a secondunnatural amino acid that is different from the first unnatural aminoacid, and where the second O-tRNA recognizes a selector codon in thenucleic acid that is different from the selector codon recognized by thefirst O-tRNA.

The methods for producing a protein with an unnatural amino acid areconducted in the context of a host cell. In these embodiments, the hostcell comprises the unnatural amino acid, the O-RS, the O-tRNA and thenucleic acid, and where culturing the host cell results in incorporatingthe unnatural amino acid. In some embodiments, the providing stepcomprises providing a eubacterial host cell (e.g., E. coli). In someembodiments, the providing step includes providing a host cell thatcontains a polynucleotide encoding the O-RS. For example, thepolynucleotide encoding the O-RS can comprise a nucleotide sequence ofSEQ ED NO: 11. In some embodiments, the step of providing a translationsystem is accomplished by providing a cell extract.

The invention also provides a variety of compositions, including nucleicacids and proteins. The nature of the composition is not particularlylimited, other than the composition comprises the specified nucleic acidor protein. The compositions of the invention can comprise any number ofadditional components of any nature.

For example, the invention provides compositions comprising O-RSpolypeptides, where the polypeptides comprise SEQ ID NO: 7-10, 12, 14,16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 50,52-55, 57, 59-63, or a conservative variant thereof, where theconservative variant polypeptide aminoacylates a cognate orthogonal tRNA(O-tRNA) with an unnatural amino acid with an efficiency that is atleast 50% of the efficiency observed for a translation system comprisingthe O-tRNA, the unnatural amino acid, and an aminoacyl-tRNA synthetasecomprising an amino acid sequence selected from SEQ ID NOs: 7-10, 12,14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 50,52-55, 57 and 59-63. The invention also provides polynucleotides thatencode any of these polypeptides above. In some embodiments, thesepolynucleotides can comprise SEQ ID NO: 11, 13, 15, 17, 19, 21, 23, 25,27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 51, 56 and 58. In someembodiments, the polypeptides are in a cell.

The invention also provides polynucleotide compositions comprising anucleotide sequence of SEQ ID NO: 11, 13, 15, 17, 19, 21, 23, 25, 27,29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 51, 56 or 58. In someembodiments, the invention provides vectors comprising thepolynucleotides, e.g., expression vectors. In some embodiments, theinvention provides cells comprising a vector described above.

DEFINITIONS

Before describing the invention in detail, it is to be understood thatthis invention is not limited to particular biological systems, whichcan, of course, vary. It is also to be understood that the terminologyused herein is for the purpose of describing particular embodimentsonly, and is not intended to be limiting. As used in this specificationand the appended claims, the singular forms “a”, “an” and “the” includeplural referents unless the content clearly dictates otherwise. Thus,for example, reference to “a cell” includes combinations of two or morecells; reference to “a polynucleotide” includes, as a practical matter,many copies of that polynucleotide.

Unless defined herein and below in the reminder of the specification,all technical and scientific terms used herein have the same meaning ascommonly understood by one of ordinary skill in the art to which theinvention pertains.

Orthogonal: As used herein, the term “orthogonal” refers to a molecule(e.g., an orthogonal tRNA (O-tRNA) and/or an orthogonal aminoacyl-tRNAsynthetase (O-RS)) that functions with endogenous components of a cellwith reduced efficiency as compared to a corresponding molecule that isendogenous to the cell or translation system, or that fails to functionwith endogenous components of the cell. In the context of tRNAs andaminoacyl-tRNA synthetases, orthogonal refers to an inability or reducedefficiency, e.g., less than 20% efficiency, less than 10% efficiency,less than 5% efficiency, or less than 1% efficiency, of an orthogonaltRNA to function with an endogenous tRNA synthetase compared to anendogenous tRNA to function with the endogenous tRNA synthetase, or ofan orthogonal aminoacyl-tRNA synthetase to function with an endogenoustRNA compared to an endogenous tRNA synthetase to function with theendogenous tRNA. The orthogonal molecule lacks a functionally normalendogenous complementary molecule in the cell. For example, anorthogonal tRNA in a cell is aminoacylated by any endogenous RS of thecell with reduced or even zero efficiency, when compared toaminoacylation of an endogenous tRNA by the endogenous RS. In anotherexample, an orthogonal RS aminoacylates any endogenous tRNA a cell ofinterest with reduced or even zero efficiency, as compared toaminoacylation of the endogenous tRNA by an endogenous RS. A secondorthogonal molecule can be introduced into the cell that functions withthe first orthogonal molecule. For example, an orthogonal tRNA/RS pairincludes introduced complementary components that function together inthe cell with an efficiency (e.g., 45% efficiency, 50% efficiency, 60%efficiency, 70% efficiency, 75% efficiency, 80% efficiency, 90%efficiency, 95% efficiency, or 99% or more efficiency) as compared tothat of a control, e.g., a corresponding tRNA/RS endogenous pair, or anactive orthogonal pair (e.g., a tyrosyl orthogonal tRNA/RS pair).

Orthogonal Tyrosyl-tRNA: As used herein, an orthogonal tyrosyl-tRNA(tyrosyl-O-tRNA) is a tRNA that is orthogonal to a translation system ofinterest, where the tRNA is: (1) identical or substantially similar to anaturally occurring leucyl or tyrosyl-tRNA, (2) derived from a naturallyoccurring leucyl or tyrosyl-tRNA by natural or artificial mutagenesis,(3) derived by any process that takes a sequence of a wild-type ormutant leucyl or tyrosyl-tRNA sequence of (1) or (2) into account, (4)homologous to a wild-type or mutant leucyl or tyrosyl-tRNA; (5)homologous to any example tRNA that is designated as a substrate for aleucyl or tyrosyl-tRNA synthetase in Table 5, or (6) a conservativevariant of any example tRNA that is designated as a substrate for aleucyl or tyrosyl-tRNA synthetase in Table 5. The leucyl or tyrosyl-tRNAcan exist charged with an amino acid, or in an uncharged state. It isalso to be understood that a “tyrosyl-O-tRNA” or “leucyl-O-tRNA”optionally is charged (aminoacylated) by a cognate synthetase with anamino acid other than tyrosine or leucine, respectively, e.g., with anunnatural amino acid. Indeed, it will be appreciated that a leucyl ortyrosyl-O-tRNA of the invention is advantageously used to insertessentially any amino acid, whether natural or artificial, into agrowing polypeptide, during translation, in response to a selectorcodon.

Orthogonal Tyrosyl Amino Acid Synthetase: As used herein, an orthogonaltyrosyl amino acid synthetase (tyrosyl-O-RS) is an enzyme thatpreferentially aminoacylates the tyrosyl-O-tRNA with an amino acid in atranslation system of interest. The amino acid that the tyrosyl-O-RSloads onto the tyrosyl-O-tRNA can be any amino acid, whether natural,unnatural or artificial, and is not limited herein. The synthetase isoptionally the same as or homologous to a naturally occurring tyrosylamino acid synthetase, or the same as or homologous to a synthetasedesignated as an O-RS in Table 5. For example, the O-RS can be aconservative variant of a tyrosyl-O-RS of Table 5, and/or can be atleast 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or more identical insequence to an O-RS of Table 5.

Similarly, an orthogonal leucyl amino acid synthetase (leucyl-O-RS) isan enzyme that preferentially aminoacylates the leucyl-O-tRNA with anamino acid in a translation system of interest. The amino acid that theleucyl-O-RS loads onto the leucyl-O-tRNA can be any amino acid, whethernatural, unnatural or artificial, and is not limited herein. Thesynthetase is optionally the same as or homologous to a naturallyoccurring leucyl amino acid synthetase, or the same as or homologous toa synthetase designated as an O-RS in Table 5. For example, the O-RS canbe a conservative variant of a leucyl-O-RS of Table 5, and/or can be atleast 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or more identical insequence to an O-RS of Table 5.

Cognate: The term “cognate” refers to components that function together,e.g., an orthogonal tRNA and an orthogonal aminoacyl-tRNA synthetase.The components can also be referred to as being complementary.

Preferentially Aminoacylates: As used herein in reference to orthogonaltranslation systems, an O-RS “preferentially aminoacylates” a cognateO-tRNA when the O-RS charges the O-tRNA with an amino acid moreefficiently than it charges any endogenous tRNA in an expression system.That is, when the O-tRNA and any given endogenous tRNA are present in atranslation system in approximately equal molar ratios, the O-RS willcharge the O-tRNA more frequently than it will charge the endogenoustRNA. Preferably, the relative ratio of O-tRNA charged by the O-RS toendogenous tRNA charged by the O-RS is high, preferably resulting in theO-RS charging the O-tRNA exclusively, or nearly exclusively, when theO-tRNA and endogenous tRNA are present in equal molar concentrations inthe translation system. The relative ratio between O-tRNA and endogenoustRNA that is charged by the O-RS, when the O-tRNA and O-RS are presentat equal molar concentrations, is greater than 1:1, preferably at leastabout 2:1, more preferably 5:1, still more preferably 10:1, yet morepreferably 20:1, still more preferably 50:1, yet more preferably 75:1,still more preferably 95:1, 98:1, 99:1, 100:1, 500:1, 1, 000:1, 5, 000:1or higher.

The O-RS “preferentially aminoacylates an O-tRNA with an unnatural aminoacid” when (a) the O-RS preferentially aminoacylates the O-tRNA comparedto an endogenous tRNA, and (b) where that aminoacylation is specific forthe unnatural amino acid, as compared to aminoacylation of the O-tRNA bythe O-RS with any natural amino acid. That is, when the unnatural andnatural amino acids are present in equal molar amounts in a translationsystem comprising the O-RS and O-tRNA, the O-RS will load the O-tRNAwith the unnatural amino acid more frequently than with the naturalamino acid. Preferably, the relative ratio of O-tRNA charged with theunnatural amino acid to O-tRNA charged with the natural amino acid ishigh. More preferably, O-RS charges the O-tRNA exclusively, or nearlyexclusively, with the unnatural amino acid. The relative ratio betweencharging of the O-tRNA with the unnatural amino acid and charging of theO-tRNA with the natural amino acid, when both the natural and unnaturalamino acids are present in the translation system in equal molarconcentrations, is greater than 1:1, preferably at least about 2:1, morepreferably 5:1, still more preferably 10:1, yet more preferably 20:1,still more preferably 50:1, yet more preferably 75:1, still morepreferably 95:1, 98:1, 99:1, 100:1, 500:1, 1, 000:1, 5, 000:1 or higher.

Selector Codon: The term “selector codon” refers to codons recognized bythe O-tRNA in the translation process and not recognized by anendogenous tRNA. The O-tRNA anticodon loop recognizes the selector codonon the mRNA and incorporates its amino acid, e.g., an unnatural aminoacid, at this site in the polypeptide. Selector codons can include,e.g., nonsense codons, such as, stop codons, e.g., amber, ochre, andopal codons; four or more base codons; rare codons; codons derived fromnatural or unnatural base pairs and/or the like.

Suppressor tRNA: A suppressor tRNA is a tRNA that alters the reading ofa messenger RNA (mRNA) in a given translation system, e.g., by providinga mechanism for incorporating an amino acid into a polypeptide chain inresponse to a selector codon. For example, a suppressor tRNA can readthrough, e.g., a stop codon (e.g., an amber, ocher or opal codon), afour base codon, a rare codon, etc.

Suppression Activity: As used herein, the term “suppression activity”refers, in general, to the ability of a tRNA (e.g., a suppressor tRNA)to allow translational read-through of a codon (e.g. a selector codonthat is an amber codon or a 4- or -more base codon) that would otherwiseresult in the termination of translation or mistranslation (e.g.,frame-shifting). Suppression activity of a suppressor tRNA can beexpressed as a percentage of translational read-through activityobserved compared to a second suppressor tRNA, or as compared to acontrol system, e.g., a control system lacking an O-RS.

The present invention provides various methods by which suppressionactivity can be quantitated. Percent suppression of a particular O-tRNAand O-RS against a selector codon (e.g., an amber codon) of interestrefers to the percentage of activity of a given expressed test marker(e.g., LacZ), that includes a selector codon, in a nucleic acid encodingthe expressed test marker, in a translation system of interest, wherethe translation system of interest includes an O-RS and an O-tRNA, ascompared to a positive control construct, where the positive controllacks the O-tRNA, the O-RS and the selector codon. Thus, for example, ifan active positive control marker construct that lacks a selector codonhas an observed activity of X in a given translation system, in unitsrelevant to the marker assay at issue, then percent suppression of atest construct comprising the selector codon is the percentage of X thatthe test marker construct displays under essentially the sameenvironmental conditions as the positive control marker was expressedunder, except that the test marker construct is expressed in atranslation system that also includes the O-tRNA and the O-RS.Typically, the translation system expressing the test marker alsoincludes an amino acid that is recognized by the O-RS and O-tRNA.Optionally, the percent suppression measurement can be refined bycomparison of the test marker to a “background” or “negative” controlmarker construct, which includes the same selector codon as the testmarker, but in a system that does not include the O-tRNA, O-RS and/orrelevant amino acid recognized by the O-tRNA and/or O-RS. This negativecontrol is useful in normalizing percent suppression measurements toaccount for background signal effects from the marker in the translationsystem of interest.

Suppression efficiency can be determined by any of a number of assaysknown in the art. For example, a β-galactosidase reporter assay can beused, e.g., a derivatived lacZ plasmid (where the construct has aselector codon n the lacZ nucleic acid sequence) is introduced intocells from an appropriate organism (e.g., an organism where theorthogonal components can be used) along with plasmid comprising anO-tRNA of the invention. A cognate synthetase can also be introduced(either as a polypeptide or a polynucleotide that encodes the cognatesynthetase when expressed). The cells are grown in media to a desireddensity, e.g., to an OD₆₀₀ of about 0.5, and β-galactosidase assays areperformed, e.g., using the BetaFluor™ β-Galactosidase Assay Kit(Novagen). Percent suppression can be calculated as the percentage ofactivity for a sample relative to a comparable control, e.g., the valueobserved from the derivatized lacZ construct, where the construct has acorresponding sense codon at desired position rather than a selectorcodon.

Translation System: The term “translation system” refers to thecomponents that incorporate an amino acid into a growing polypeptidechain (protein). Components of a translation system can include, e.g.,ribosomes, tRNAs, synthetases, mRNA and the like. The O-tRNA and/or theO-RSs of the invention can be added to or be part of an in vitro or invivo translation system, e.g., in a non-eukaryotic cell, e.g., abacterium (such as E. coli), or in a eukaryotic cell, e.g., a yeastcell, a mammalian cell, a plant cell, an algae cell, a fungus cell, aninsect cell, and/or the like.

Unnatural Amino Acid: As used herein, the term “unnatural amino acid”refers to any amino acid, modified amino acid, and/or amino acidanalogue, that is not one of the 20 common naturally occurring aminoacids or seleno cysteine or pyrrolysine. For example, FIG. 1 provides 17unnatural amino acids that find use with the invention.

Derived from: As used herein, the term “derived from” refers to acomponent that is isolated from or made using a specified molecule ororganism, or information from the specified molecule or organism. Forexample, a polypeptide that is derived from a second polypeptidecaninclude an amino acid sequence that is identical or substantiallysimilar to the amino acid sequence of the second polypeptide. In thecase of polypeptides, the derived species can be obtained by, forexample, naturally occurring mutagenesis, artificial directedmutagenesis or artificial random mutagenesis. The mutagenesis used toderive polypeptides can be intentionally directed or intentionallyrandom, or a mixture of each. The mutagenesis of a polypepitde to createa different polypeptide derived from the first can be a random event(e.g., caused by polymerase infidelity) and the identification of thederived polypeptide can be made by appropriate screening methods, e.g.,as discussed herein. Mutagenesis of a polypeptide typically entailsmanipulation of the polynucleotide that encodes the polypeptide.

Positive Selection or Screening Marker: As used herein, the term“positive selection or screening marker” refers to a marker that, whenpresent, e.g., expressed, activated or the like, results inidentification of a cell, which comprises the trait, e.g., a cell withthe positive selection marker, from those without the trait.

Negative Selection or Screening Marker: As used herein, the term“negative selection or screening marker” refers to a marker that, whenpresent, e.g., expressed, activated, or the like, allows identificationof a cell that does not comprise a selected property or trait (e.g., ascompared to a cell that does possess the property or trait).

Reporter: As used herein, the term “reporter” refers to a component thatcan be used to identify and/or select target components of a system ofinterest. For example, a reporter can include a protein, e.g., anenzyme, that confers antibiotic resistance or sensitivity (e.g.,β-lactamase, chloramphenicol acetyltransferase (CAT), and the like), afluorescent screening marker (e.g., green fluorescent protein (e.g.,(GFP), YFP, EGFP, RFP, etc.), a luminescent marker (e.g., a fireflyluciferase protein), an affinity based screening marker, or positive ornegative selectable marker genes such as lacZ, β-gal/lacZ(β-galactosidase), ADH (alcohol dehydrogenase), his3, ura3, leu2, lys2,or the like.

Eukaryote: As used herein, the term “eukaryote” refers to organismsbelonging to the Kingdom Eucarya. Eukaryotes are generallydistinguishable from prokaryotes by their typically multicellularorganization (but not exclusively multicellular, for example, yeast),the presence of a membrane-bound nucleus and other membrane-boundorganelles, linear genetic material (i.e., linear chromosomes), theabsence of operons, the presence of introns, message capping and poly-AmRNA, and other biochemical characteristics, such as a distinguishingribosomal structure. Eukaryotic organisms include, for example, animals(e.g., mammals, insects, reptiles, birds, etc.), ciliates, plants (e.g.,monocots, dicots, algae, etc.), fungi, yeasts, flagellates,microsporidia, protists, etc.

Prokaryote: As used herein, the term “prokaryote” refers to organismsbelonging to the Kingdom Monera (also termed Procarya). Prokaryoticorganisms are generally distinguishable from eukaryotes by theirunicellular organization, asexual reproduction by budding or fission,the lack of a membrane-bound nucleus or other membrane-bound organelles,a circular chromosome, the presence of operons, the absence of introns,message capping and poly-A mRNA, and other biochemical characteristics,such as a distinguishing ribosomal structure. The Prokarya includesubkingdoms Eubacteria and Archaea (sometimes termed “Archaebacteria”).Cyanobacteria (the blue green algae) and mycoplasma are sometimes givenseparate classifications under the Kingdom Monera.

Bacteria: As used herein, the terms “bacteria” and “eubacteria” refer toprokaryotic organisms that are distinguishable from Archaea. Similarly,Archaea refers to prokaryotes that are distinguishable from eubacteria.Eubacteria and Archaea can be distinguished by a number morphologicaland biochemical criteria. For example, differences in ribosomal RNAsequences, RNA polymerase structure, the presence or absence of introns,antibiotic sensitivity, the presence or absence of cell wallpeptidoglycans adn other cell wall components, the branched versusunbranched structures of membrane lipids, and the presence/absence ofhistones and histone-like proteins are used to assign an organism toEubacteria or Archaea.

Examples of Eubacteria include Escherichia coli, Thermus thermophilusand Bacillus stearothermophilus. Example of Archaea includeMethanococcus jannaschii (Mj), Methanosarcina mazei (Mm),Methanobacteriurn thermoautotrophicum (Mt), Methanococcus maripaludis,Methanopyrus kandleri, Halobacterium such as Haloferax volcanii andHalobacterium species NRC-1, Archaeoglobus fulgidus (Af), Pyrococcusfuriosus (Pf), Pyrococcus horikoshii (Ph), Pyrobaculum aerophilum,Pyrococcus abyssi, Sulfolobus solfataricus (Ss), Sulfolobus tokodaii,Aeuropyrum pernix (Ap), Thermoplasma acidophilum and Thermoplasmavolcanium.

Conservative Variant: As used herein, the term “conservative variant,”in the context of a translation component, refers to a translationcomponent, e.g., a conservative variant O-tRNA or a conservative variantO-RS, that functionally performs similar to a base component that theconservative variant is similar to, e.g., an O-tRNA or O-RS, havingvariations in the sequence as compared to a reference O-tRNA or O-RS.For example, an O-RS, or a conservative variant of that O-RS, willaminoacylate a cognate O-tRNA with an unnatural amino acid, e.g., anamino acid comprising an N-acetylgalactosamine moiety. In this example,the O-RS and the conservative variant O-RS do not have the same aminoacid sequences. The conservative variant can have, e.g., one variation,two variations, three variations, four variations, or five or morevariations in sequence, as long as the conservative variant is stillcomplementary to the corresponding O-tRNA or O-RS.

In some embodiments, a conservative variant O-RS comprises one or moreconservative amino acid substitutions compared to the O-RS from which itwas derived. In some embodiments, a conservative variant O-RS comprisesone or more conservative amino acid substitutions compared to the O-RSfrom which it was derived, and furthermore, retains O-RS biologicalactivity; for example, a conservative variant O-RS that retains at least10% of the biological activity of the parent O-RS molecule from which itwas derived, or alternatively, at least 20%, at least 30%, or at least40%. In some preferred embodiments, the conservative variant O-RSretains at least 50% of the biological activity of the parent O-RSmolecule from which it was derived. The conservative amino acidsubstitutions of a conservative variant O-RS can occur in any domain ofthe O-RS, including the amino acid binding pocket.

Selection or Screening Agent: As used herein, the term “selection orscreening agent” refers to an agent that, when present, allows forselection/screening of certain components from a population. Forexample, a selection or screening agent can be, but is not limited to,e.g., a nutrient, an antibiotic, a wavelength of light, an antibody, anexpressed polynucleotide, or the like. The selection agent can bevaried, e.g., by concentration, intensity, etc.

In Response to: As used herein, the term “in response to” refers to theprocess in which an O-tRNA of the invention recognizes a selector codonand mediates the incorporation of the unnatural amino acid, which iscoupled to the tRNA, into the growing polypeptide chain.

Encode: As used herein, the term “encode” refers to any process wherebythe information in a polymeric macromolecule or sequence string is usedto direct the production of a second molecule or sequence string that isdifferent from the first molecule or sequence string. As used herein,the term is used broadly, and can have a variety of applications. Insome aspects, the term “encode” describes the process ofsemi-conservative DNA replication, where one strand of a double-strandedDNA molecule is used as a template to encode a newly synthesizedcomplementary sister strand by a DNA-dependent DNA polymerase.

In another aspect, the term “encode” refers to any process whereby theinformation in one molecule is used to direct the production of a secondmolecule that has a different chemical nature from the first molecule.For example, a DNA molecule can encode an RNA molecule (e.g., by theprocess of transcription incorporating a DNA-dependent RNA polymeraseenzyme). Also, an RNA molecule can encode a polypeptide, as in theprocess of translation. When used to describe the process oftranslation, the term “encode” also extends to the triplet codon thatencodes an amino acid. In some aspects, an RNA molecule can encode a DNAmolecule, e.g., by the process of reverse transcription incorporating anRNA-dependent DNA polymerase. In another aspect, a DNA molecule canencode a polypeptide, where it is understood that “encode” as used inthat case incorporates both the processes of transcription andtranslation.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 provides the chemical structures of various unnatural aminoacids.

FIG. 2 provides a photograph of a stained SDS-PAGE analysis of theZ-domain protein accumulated in the presence (lane 2) or absence (lane3) of p-nitro-L-phenylalanine. Lane 1 contains molecular mass markers.

FIG. 3 provides a MALDI-TOF analysis of p-nitro-L-phenylalanineincorporated Z-domain protein. Expected mass: 7958, 7826 (exclusion offirst methionine); observed: 7958, 7828.

FIG. 4A provides a fluorescence spectra of the ²²Trp GCN4pl mutant(solid line) and the mixture of ²²Trp and ²²p-nitro-L-phenylalanineGCN4pl mutants (broken line).

FIG. 4B provides a fluorescence spectra of the ⁵⁵Trp GCN4pl mutant(solid line) and the mixture of ⁵⁵Trp and ²²p-nitro-L-phenylalanineGCN4pl mutants (broken line).

FIG. 5A provides the chemical structure of 1,5-dansylalanine. FIG. 5Bprovides a model of the Thermus thermophilus leucyl-tRNA synthetase(LRS) active site with bound 1,5-dansylalanine-AMP-amide. Active siteresidues of mutant LRS clone B8 that are part of the randomized regionare shown as sticks. The numbering corresponds to E. coli LRS.

FIGS. 6A and 6B describe the redesign strategy of the mutant B8leucyl-tRNA synthetase editing site. FIG. 6A provides the crystalstructure of the editing site of Therrnus thermophilus leucyl-tRNAsynthetase in complex with 2′-(L-norvalyl)-amino-2′-deoxyadenosinemimicking the charged tRNA 3′-terminus. T252 and V340 are shown assticks. FIG. 6B provides SDS-PAGE analysis of Ni-NTA purified hSODbearing dansylalanine at position 33 using leucyl-tRNA synthetase cloneB8 and the two mutants V338A and T252A. The upper gel is a photograph ofa Coomassie stain. The lower gel is a fluorescence image with excitationat 302 nm and emission detection at 520 nm. L=molecular weight ladder;UAA=unnatural amino acid.

FIGS. 7A and 7B describe the enhanced amber suppression efficiency of E.coli leucyl-tRNA synthetase clone G2-6, generated by error prone PCR andselection. FIG. 7A provides the crystal structure of Thermusthermophilus leucyl-tRNA synthetase (Cusack et al., EMBO J.,19(10):2351-2361 [2000]). The synthetic domain, editing domain, aminoacids randomized in the homologous E. coli synthetase, and amino acidschanged by error prone PCR present in the G2-6 clone are all indicated.FIG. 7B provides a Coomassie stained SDS-PAGE analysis of expressed hSODbearing o-nitrobenzylserine at position 33 using E. coli leucyl-tRNAmutant synthetase clone 3H11 designed for incorporation ofo-nitrobenzylcysteine and mutant E. coli leucyl-tRNA synthetase cloneG2-6 evolved for efficient suppression with o-nitrobenzylserine.L=molecular weight ladder; UAA=unnatural amino acid (oNBS).

FIG. 8 provides a schematic representation of the photodecaging(photoactivation) of the caged tyrosine moleculeO-(2-nitrobenzyl)-L-tyrosine by irradiation at 365 nm, resulting incleavage of the benzylic CO-bond and rapid formation of the decagedamino acid.

FIG. 9 provides concentration curve assays illustrating theexperimentally observed photodecaging (photoactivation) of the cagedtyrosine molecule O-(2-nitrobenzyl)-L-tyrosine. Photodecaging ofO-(2-nitrobenzyl)-L-tyrosine was illustrated by irradiation of a 0.2 mMamino acid solution in water using a handheld UV lamp (365 nm at 10 mmdistance). Aliquots were taken at specific time points and analyzed byLC/MS. The concentrations of O-(2-nitrobenzyl)-L-tyrosine (squares) andthe corresponding decaged species (circles) are shown.

FIG. 10 provides a Gelcode Blue stained SDS-PAGE of myoglobin 74TAGexpressed in the presence or absence of O-(2-nitrobenzyl)-L-tyrosineusing three different mutant synthetases.

FIGS. 11A and 11B provide an LC-MS/MS analysis of 74TAG mutant myoglobinprotein showing tyrosine at position 74 (tryptic peptideHGVTVLTALGYILK).

FIGS. 12A and 12B provide an LC-MS/MS analysis similar to that describedin FIGS. 11A and 11B, except where the analysis uses a deuteratedO-(2-nitrobenzyl)-L-tyrosine, where J denotes the deuterated amino acid(tryptic peptide HGVTVLTALGJILK).

FIG. 13 provides superposition graphs of a para-cyanophenylalanine IRspectra taken in THF and water.

FIG. 14A provides the background-subtracted spectrum ofpara-cyanophenylalanine (solid) fit to a Gaussian curve (dashed). FIG.14B provides the background-subtracted spectrum ofmeta-cyanophenylalanine (solid) fit to two Gaussian curves (dashed).

FIG. 15 provides a schematic describing the synthesis ofp-ethylthiocarbonyl-L-phenylalanine

FIG. 16 provides a MALDI-TOF mass spectrum analysis result of mutant Zdomain proteins containing unnatural amino acid at the seventh position.All the experimentally obtained mass data are in excellent agreementwith those calculated masses of intact proteins containing eitherthioester or carboxylic acid groups.

FIG. 17 provides a schematic of protein labeling by chemical ligation.

FIGS. 18A-18D provide LC/MS elution profiles (first peak: 3; secondpeak: 2) monitored at 340 nm. FIG. 18A shows a profile using a 1:1mixture of 3 and 2 in MeOH. FIG. 18B shows a profile using 3 in PBS(pH=7.4, reaction time: 1 week). FIG. 18C shows a profile using 3 in PBS(pH=3.9, reaction time: 4 days). FIG. 18D shows a profile using 3 indiluted H₂SO₄ solution (pH=1.9, reaction time: 12 hrs). All reactionswere done at room temperature with constant stirring.

FIG. 19 provides a schematic describing the synthesis of thediketone-containing unnatural amino acidp-(3-oxobutanoyl)-L-phenylalanine.

FIG. 20 provides a Gelcode Blue stained SDS-PAGE analysis of expressed Zdomain protein in the presence or absence ofp-(3-oxobutanoyl)-L-phenylalanine. The analysis shows the in vitrolabeling of mutant Z domain protein containingp-(3-oxobutanoyl)-L-phenylalanine with fluorescein hydrazide. wt=wildtype.

FIG. 21 provides a scheme describing the synthesis of various adducts ofthe diketone-containing moiety.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides solutions to the inherent limitations of using atranslation system confined by the 20 naturally occurring amino acids.The solutions include the programmed, site-specific biosyntheticincorporation of unnatural amino acids with novel properties intoproteins using orthogonal translation systems. We describe herein novelcompositions (e.g., novel aminoacyl-tRNA synthetases) and novel methodsfor the highly efficient and specific genetic incorporation of a varietyof unnatural amino acids into proteins in response to a selector codon(e.g., the amber nonsense codon, TAG).

In some cases, the unnatural amino acid side chains can then bespecifically and regioselectively modified. Because of the uniquereaction chemistries of these unnatural amino acid substituents,proteins into which they are incorporated can be modified with extremelyhigh selectivity. In some cases, the unnatural amino acid reactive grouphas the advantage of being completely alien to in vivo systems, therebyimproving reaction selectivity. In some aspects, the modificationreactions can be conducted using relatively mild reaction conditionsthat permit both in vitro and in vivo conjugation reactions involvingproteins, and preserving protein biological activity. The nature of thematerial that is conjugated to an unnatural amino acid in a protein isnot particularly limited, and can be any desired entity, e.g., dyes,fluorophores, crosslinking agents, saccharide derivatives, polymers(e.g., derivatives of polyethylene glycol), photocrosslinkers, cytotoxiccompounds, affinity labels, derivatives of biotin, resins, beads, asecond protein or polypeptide (or more), polynucleotide(s) (e.g., DNA,RNA, etc.), metal chelators, cofactors, fatty acids, carbohydrates, andthe like.

In other aspects, the incorporated unnatural amino acid imparts novelbiological properties to the protein into which it is incorporated. Forexample, the unnatural amino acid can be a fluorescent amino acid, aphotocaged or photoactivatable amino acid, an amino acid that canparticipate in a FRET pair as a donor or acceptor, a redox-active aminoacid, a metal-chelating amino acid, etc.

In some aspects, to demonstrate (but not to limit) the presentinvention, the disclosure herein demonstrates that the unnatural aminoacid moiety can be incorporated into a model protein. It is not intendedthat the incorporation of the unnatural amino acid be limited to such amodel protein. From the present disclosure, it will be clear that theincorporation of an unnatural amino acid into any given protein ofinterest is advantageous for a wide variety of proteins for use intherapeutic and research purposes.

We have evolved novel orthogonal tRNA/aminoacyl-tRNA synthetase pairsthat function in eubacteria and yeast to site specifically incorporateunnatural amino acids (e.g., the unnatural amino acids provided inFIG. 1) in response to selector codons. Briefly, we have identifiednovel mutants of the Methanococcus janaschii tyrosyl-tRNA synthetase andthe Escherichia coli leucyl-tRNA synthetase that selectively charge asuppressor tRNA with an unnatural amino acid in either E. coli hostcells or yeast host cells, respectively.

These evolved tRNA-synthetase pairs can be used to site-specificallyincorporate the respective unnatural amino acid into a protein. Theincorporation of the unnatural amino acid into the protein can beprogrammed to occur at any desired position by engineering thepolynucleotide encoding the protein of interest to contain a selectorcodon that signals the incorporation of the unnatural amino acid.

Orthogonal tRNA/Aminoacyl-tRNA Synthetase Technology

An understanding of the novel compositions and methods of the presentinvention is facilitated by an understanding of the activitiesassociated with orthogonal tRNA and orthogonal aminoacyl-tRNA synthetasepairs. Discussions of orthogonal tRNA and aminoacyl-tRNA synthetasetechnologies can be found, for example, in International Publications WO2002/085923, WO 2002/086075, WO 204/09459, WO 2005/019415, WO2005/007870 and WO 2005/007624. See also, Wang and Schultz “Expandingthe Genetic Code,” Angewandte Chemie Int. Ed., 44(1):34-66 (2005), thecontent of which is incorporated by reference in its entirety.

In order to add additional reactive unnatural amino acids to the geneticcode, new orthogonal pairs comprising an aminoacyl-tRNA synthetase and asuitable tRNA are needed that can function efficiently in the hosttranslational machinery, but that are “orthogonal” to the translationsystem at issue, meaning that it functions independently of thesynthetases and tRNAs endogenous to the translation system. Desiredcharacteristics of the orthologous pair include tRNA that decode orrecognize only a specific codon, e.g., a selector codon, that is notdecoded by any endogenous tRNA, and aminoacyl-tRNA synthetases thatpreferentially aminoacylate (or “charge”) its cognate tRNA with only onespecific unnatural amino acid. The O-tRNA is also not typicallyaminoacylated by endogenous synthetases. For example, in E. coli, anorthogonal pair will include an aminoacyl-tRNA synthetase that does notcross-react with any of the endogenous tRNA, e.g., which there are 40 inE. colti, and an orthogonal tRNA that is not aminoacylated by any of theendogenous synthetases, e.g., of which there are 21 in E. coli.

The invention described herein provides orthogonal pairs for the geneticencoding and incorporation of unnatural amino acids into proteins in aeubacteria, e.g., an E. coli, or in yeast, where the orthogonalcomponents do not cross-react with endogenous E. coli or yeastcomponents of the translational machinery of the host cell, butrecognize the desired unnatural amino acid and incorporate it intoproteins in response to the selector codon (e.g., an amber nonsensecodon, TAG). The orthogonal components provided by the invention includeorthogonal aminoacyl-tRNA synthetases derived from Methanococcusjannaschii tyrosyl tRNA-synthetase, and the mutant tyrosyl tRNA_(CUA)amber suppressor, which function as an orthogonal pair in a eubacterialhost cell. The invention also provides orthogonal components derivedfrom E. coli leucyl-tRNA-synthetase, and a mutant E. coli leucyltRNA_(CUA) amber suppressor, which function as an orthogonal pair in ayeast host cell. In these systems, the mutant aminoacyl-tRNA synthetasesaminoacylate the suppressor tRNA with its respective unnatural aminoacid and not with any of the common twenty amino acids.

This invention provides compositions of and methods for identifying andproducing additional orthogonal tRNA-aminoacyl-tRNA synthetase pairs,e.g., O-tRNA/O-RS pairs that can be used to incorporate an unnaturalamino acid into a protein. An O-tRNA/O-RS pair of the invention iscapable of mediating incorporation of an unnatural amino acid, forexample, an unnatural amino acid shown in FIG. 1, into a protein that isencoded by a polynucleotide, where the polynucleotide comprises aselector codon that is recognized by the O-tRNA, e.g., in vivo. Theanticodon loop of the O-tRNA recognizes the selector codon on an mRNAand incorporates its amino acid, e.g., an unnatural amino acid shown inFIG. 1, at this site in the polypeptide. Generally, an orthogonalaminoacyl-tRNA synthetase of the invention preferentially aminoacylates(or charges) its O-tRNA with only one specific unnatural amino acid.

The ability to incorporate an unnatural amino acid (e.g., an unnaturalamino acid provided in FIG. 1) site-specifically into proteins canfacilitate the study of proteins, as well as enable the engineering ofproteins with novel properties. For example, expression of proteinscontaining one or more unnatural amino acids can facilitate the study ofproteins by specific labeling, alter catalytic function of enzymes,improve, biological activity or reduce cross-reactivity to a substrate,crosslink a protein with other proteins, small molecules orbiomolecules, reduce or eliminate protein degradation, improve half-lifeof proteins in vivo (e.g., by pegylation or other modifications ofintroduced reactive sites), etc.

Orthogonal tRNA/Orthogonal Aminoacyl-tRNA Synthetases and Pairs Thereof

Translation systems that are suitable for making proteins that includeone or more unnatural amino acid are generally described in, forexample, International Publication Numbers WO 2002/086075, entitled“METHODS AND COMPOSITION FOR THE PRODUCTION OF ORTHOGONALtRNA-AMINOACYL-tRNA SYNTHETASE PAIRS;” WO 2002/085923, entitled “IN VIVOINCORPORATION OF UNNATURAL AMINO ACIDS;” and WO 2004/094593, entitled“EXPANDING THE EUKARYOTIC GENETIC CODE;” WO 2005/019415, filed Jul. 7,2004; WO 2005/007870, filed Jul. 7, 2004 and WO 2005/007624, filed Jul.7, 2004. Each of these applications is incorporated herein by referencein its entirety. See also, Wang and Schultz “Expanding the GeneticCode,” Angewandte Chemie Int. Ed., 44(1):34-66 (2005), the content ofwhich is incorporated by reference in its entirety. Such translationsystems generally comprise cells (which can be non-eukaryotic cells suchas E. coli, or eukaryotic cells such as yeast) that include anorthogonal tRNA (O-tRNA), an orthogonal aminoacyl tRNA synthetase(O-RS), and an unnatural amino acid, where the O-RS aminoacylates theO-tRNA with the unnatural amino acid. An orthogonal pair of theinvention includes an O-tRNA, e.g., a suppressor tRNA, a frameshifttRNA, or the like, and an O-RS. Individual components are also providedin the invention.

In general, when an orthogonal pair recognizes a selector codon andloads an amino acid in response to the selector codon, the orthogonalpair is said to “suppress” the selector codon. That is, a selector codonthat is not recognized by the translation system's (e.g., the cell's)endogenous machinery is not ordinarily translated, which can result inblocking production of a polypeptide that would otherwise be translatedfrom the nucleic acid. An O-tRNA of the invention recognizes a selectorcodon and includes at least about, e.g., a 45%, a 50%, a 60%, a 75%, a80%, or a 90% or more suppression efficiency in the presence of acognate synthetase in response to a selector codon as compared to thesuppression efficiency of an O-tRNA comprising or encoded by apolynucleotide sequence as set forth in the sequence listing herein. TheO-RS aminoacylates the O-tRNA with an unnatural amino acid of interest.The cell uses the O-tRNA/O-RS pair to incorporate the unnatural aminoacid into a growing polypeptide chain, e.g., via a nucleic acid thatcomprises a polynucleotide that encodes a polypeptide of interest, wherethe polynucleotide comprises a selector codon that is recognized by theO-tRNA. In certain desirable aspects, the cell can include an additionalO-tRNA/O-RS pair, where the additional O-tRNA is loaded by theadditional O-RS with a different unnatural amino acid. For example, oneof the O-tRNAs can recognize a four base codon and the other canrecognize a stop codon. Alternately, multiple different stop codons ormultiple different four base codons can specifically recognize differentselector codons.

In certain embodiments of the invention, a cell such as an E. coli cellor a yeast cell that includes an orthogonal tRNA (O-tRNA), an orthogonalaminoacyl-tRNA synthetase (O-RS), an unnatural amino acid and a nucleicacid that comprises a polynucleotide that encodes a polypeptide ofinterest, where the polynucleotide comprises the selector codon that isrecognized by the O-tRNA. The translation system can also be a cell-freesystem, e.g., any of a variety of commercially available “in vitro”transcription/translation systems in combination with an O-tRNA/ORS pairand an unnatural amino acid as described herein.

In one embodiment, the suppression efficiency of the O-RS and the O-tRNAtogether is about, e.g., 5 fold, 10 fold, 15 fold, 20 fold, or 25 foldor more greater than the suppression efficiency of the O-tRNA lackingthe O-RS. In some aspect, the suppression efficiency of the O-RS and theO-tRNA together is at least about, e.g., 35%, 40%, 45%, 50%, 60%, 75%,80%, or 90% or more of the suppression efficiency of an orthogonalsynthetase pair as set forth in the sequence listings herein.

As noted, the invention optionally includes multiple O-tRNA/O-RS pairsin a cell or other translation system, which allows incorporation ofmore than one unnatural amino acid. For example, the cell can furtherinclude an additional different O-tRNA/O-RS pair and a second unnaturalamino acid, where this additional O-tRNA recognizes a second selectorcodon and this additional O-RS preferentially aminoacylates the O-tRNAwith the second unnatural amino acid. For example, a cell that includesan O-tRNA/O-RS pair (where the O-tRNA recognizes, e.g., an amberselector codon), can further comprise a second orthogonal pair, wherethe second O-tRNA recognizes a different selector codon, e.g., an opalcodon, a four-base codon, or the like. Desirably, the differentorthogonal pairs are derived from different sources, which canfacilitate recognition of different selector codons.

The O-tRNA and/or the O-RS can be naturally occurring or can be, e.g.,derived by mutation of a naturally occurring tRNA and/or RS, e.g., bygenerating libraries of tRNAs and/or libraries of RSs, from any of avariety of organisms and/or by using any of a variety of availablemutation strategies. For example, one strategy for producing anorthogonal tRNA/aminoacyl-tRNA synthetase pair involves importing aheterologous (to the host cell) tRNA/synthetase pair from, e.g., asource other than the host cell, or multiple sources, into the hostcell. The properties of the heterologous synthetase candidate include,e.g., that it does not charge any host cell tRNA, and the properties ofthe heterologous tRNA candidate include, e.g., that it is notaminoacylated by any host cell synthetase. In addition, the heterologoustRNA is orthogonal to all host cell synthetases.

A second strategy for generating an orthogonal pair involves generatingmutant libraries from which to screen and/or select an O-tRNA or O-RS.These strategies can also be combined.

Orthogonal tRNA (O-tRNA)

An orthogonal tRNA (O-tRNA) of the invention desirably mediatesincorporation of an unnatural amino acid into a protein that is encodedby a polynucleotide that comprises a selector codon that is recognizedby the O-tRNA, e.g., in vivo or in vitro. In certain embodiments, anO-tRNA of the invention includes at least about, e.g., a 45%, a 50%, a60%, a 75%, a 80%, or a 90% or more suppression efficiency in thepresence of a cognate synthetase in response to a selector codon ascompared to an O-tRNA comprising or encoded by a polynucleotide sequenceas set forth in the O-tRNA sequences in the sequence listing herein.

Suppression efficiency can be determined by any of a number of assaysknown in the art. For example, a β-galactosidase reporter assay can beused, e.g., a derivatized lacZ plasmid (where the construct has aselector codon n the lacZ nucleic acid sequence) is introduced intocells from an appropriate organism (e.g., an organism where theorthogonal components can be used) along with plasmid comprising anO-tRNA of the invention. A cognate synthetase can also be introduced(either as a polypeptide or a polynucleotide that encodes the cognatesynthetase when expressed). The cells are grown in media to a desireddensity, e.g., to an OD₆₀₀ of about 0.5, and β-galactosidase assays areperformed, e.g., using the BetaFluor™ β-Galactosidase Assay Kit(Novagen). Percent suppression can be calculated as the percentage ofactivity for a sample relative to a comparable control, e.g., the valueobserved from the derivatized lacZ construct, where the construct has acorresponding sense codon at desired position rather than a selectorcodon.

Examples of O-tRNAs of the invention are set forth in the sequencelisting herein. See also, the tables, examples and figures herein forsequences of exemplary O-tRNA and O-RS molecules. See also, the sectionentitled “Nucleic Acid and Polypeptide Sequence and Variants” herein. Inan RNA molecule, such as an O-RS mRNA, or O-tRNA molecule, Thymine (T)is replace with Uracil (U) relative to a given sequence (or vice versafor a coding DNA), or complement thereof. Additional modifications tothe bases can also be present.

The invention also includes conservative variations of O-tRNAscorresponding to particular O-tRNAs herein. For example, conservativevariations of O-tRNA include those molecules that function like theparticular O-tRNAs, e.g., as in the sequence listing herein and thatmaintain the tRNA L-shaped structure by virtue of appropriateself-complementarity, but that do not have a sequence identical tothose, e.g., in the sequence listing, figures or examples herein (and,desirably, are other than wild type tRNA molecules). See also, thesection herein entitled “Nucleic acids and Polypeptides Sequence andVariants.”

The composition comprising an O-tRNA can further include an orthogonalaminoacyl-tRNA synthetase (O-RS), where the O-RS preferentiallyaminoacylates the O-tRNA with an unnatural amino acid. In certainembodiments, a composition including an O-tRNA can further include atranslation system (e.g., in vitro or in vivo). A nucleic acid thatcomprises a polynucleotide that encodes a polypeptide of interest, wherethe polynucleotide comprises a selector codon that is recognized by theO-tRNA, or a combination of one or more of these can also be present inthe cell. See also, the section herein entitled “Orthogonalaminoacyl-tRNA synthetases.”

Methods of producing an orthogonal tRNA (O-tRNA) are also a feature ofthe invention. An O-tRNA produced by the method is also a feature of theinvention. In certain embodiments of the invention, the O-tRNAs can beproduced by generating a library of mutants. The library of mutant tRNAscan be generated using various mutagenesis techniques known in the art.For example, the mutant tRNAs can be generated by site-specificmutations, random point mutations, homologous recombination, DNAshuffling or other recursive mutagenesis methods, chimeric constructionor any combination thereof, e.g., of the example O-tRNA of Table 5.

Additional mutations can be introduced at a specific position(s), e.g.,at a nonconservative position(s), or at a conservative position, at arandomized position(s), or a combination of both in a desired loop orregion of a tRNA, e.g., an anticodon loop, the acceptor stem, D arm orloop, variable loop, TPC arm or loop, other regions of the tRNAmolecule, or a combination thereof. Typically, mutations in a tRNAinclude mutating the anticodon loop of each member of the library ofmutant tRNAs to allow recognition of a selector codon. The method canfurther include adding additional sequences to the O-tRNA. Typically, anO-tRNA possesses an improvement of orthogonality for a desired organismcompared to the starting material, e.g., the plurality of tRNAsequences, while preserving its affinity towards a desired RS.

The methods optionally include analyzing the similarity (and/or inferredhomology) of sequences of tRNAs and/or aminoacyl-tRNA synthetases todetermine potential candidates for an O-tRNA, O-RS and/or pairs thereof,that appear to be orthogonal for a specific organism. Computer programsknown in the art and described herein can be used for the analysis,e.g., BLAST and pileup programs can be used. In one example, to choosepotential orthogonal translational components for use in E. coli, asynthetase and/or a tRNA is chosen that does not display close sequencesimilarity to eubacterial organisms.

Typically, an O-tRNA is obtained by subjecting to, e.g., negativeselection, a population of cells of a first species, where the cellscomprise a member of the plurality of potential O-tRNAs. The negativeselection eliminates cells that comprise a member of the library ofpotential O-tRNAs that is aminoacylated by an aminoacyl-tRNA synthetase(RS) that is endogenous to the cell. This provides a pool of tRNAs thatare orthogonal to the cell of the first species.

In certain embodiments, in the negative selection, a selector codon(s)is introduced into a polynucleotide that encodes a negative selectionmarker, e.g., an enzyme that confers antibiotic resistance, e.g.,β-lactamase, an enzyme that confers a detectable product, e.g.,β-galactosidase, chloramphenicol acetyltransferase (CAT), e.g., a toxicproduct, such as barnase, at a nonessential position (e.g., stillproducing a functional barnase), etc. Screening/selection is optionallydone by growing the population of cells in the presence of a selectiveagent (e.g., an antibiotic, such as ampicillin). In one embodiment, theconcentration of the selection agent is varied.

For example, to measure the activity of suppressor tRNAs, a selectionsystem is used that is based on the in vivo suppression of selectorcodon, e.g., nonsense (e.g., stop) or framcshift mutations introducedinto a polynucleotide that encodes a negative selection marker, e.g., agene for β-lactamase (bla). For example, polynucleotide variants, e.g.,bla variants, with a selector codon at a certain position (e.g., A184),are constructed. Cells, e.g., bacteria, are transformed with thesepolynucleotides. In the case of an orthogonal tRNA, which cannot beefficiently charged by endogenous E. coli synthetases, antibioticresistance, e.g., ampicillin resistance, should be about or less thanthat for a bacteria transformed with no plasmid. If the tRNA is notorthogonal, or if a heterologous synthetase capable of charging the tRNAis co-expressed in the system, a higher level of antibiotic, e.g.,ampicillin, resistance is be observed. Cells, e.g., bacteria, are chosenthat are unable to grow on LB agar plates with antibiotic concentrationsabout equal to cells transformed with no plasmids.

In the case of a toxic product (e.g., ribonuclease or barnase), when amember of the plurality of potential tRNAs is aminoacylated byendogenous host, e.g., Escherichia coli synthetases (i.e., it is notorthogonal to the host, e.g., Escherichia coli synthetases), theselector codon is suppressed and the toxic polynucleotide productproduced leads to cell death. Cells harboring orthogonal tRNAs ornon-functional tRNAs survive.

In one embodiment, the pool of tRNAs that are orthogonal to a desiredorganism are then subjected to a positive selection in which a selectorcodon is placed in a positive selection marker, e.g., encoded by a drugresistance gene, such a β-lactamase gene.

The positive selection is performed on a cell comprising apolynucleotide encoding or comprising a member of the pool of tRNAs thatare orthogonal to the cell, a polynucleotide encoding a positiveselection marker, and a polynucleotide encoding a cognate RS. In certainembodiments, the second population of cells comprises cells that werenot eliminated by the negative selection. The polynucleotides areexpressed in the cell and the cell is grown in the presence of aselection agent, e.g., ampicillin. tRNAs are then selected for theirability to be aminoacylated by the coexpressed cognate synthetase and toinsert an amino acid in response to this selector codon. Typically,these cells show an enhancement in suppression efficiency compared tocells harboring non-functional tRNA(s), or tRNAs that cannot efficientlybe recognized by the synthetase of interest. The cell harboring thenon-functional tRNAs or tRNAs that are not efficiently recognized by thesynthetase of interest, are sensitive to the antibiotic. Therefore,tRNAs that: (i) are not substrates for endogenous host, e.g.,Escherichia coli, synthetases; (ii) can be aminoacylated by thesynthetase of interest; and (iii) are functional in translation, surviveboth selections.

Accordingly, the same marker can be either a positive or negativemarker, depending on the context in which it is screened. That is, themarker is a positive marker if it is screened for, but a negative markerif screened against.

The stringency of the selection, e.g., the positive selection, thenegative selection or both the positive and negative selection, in theabove described-methods, optionally includes varying the selectionstringency. For example, because barnase is an extremely toxic protein,the stringency of the negative selection can be controlled byintroducing different numbers of selector codons into the barnase geneand/or by using an inducible promoter. In another example, theconcentration of the selection or screening agent is varied (e.g.,ampicillin concentration). In some aspects of the invention, thestringency is varied because the desired activity can be low duringearly rounds. Thus, less stringent selection criteria are applied inearly rounds and more stringent criteria are applied in later rounds ofselection. In certain embodiments, the negative selection, the positiveselection or both the negative and positive selection can be repeatedmultiple times. Multiple different negative selection markers, positiveselection markers or both negative and positive selection markers can beused. In certain embodiments, the positive and negative selection markercan be the same.

Other types of selections/screening can be used in the invention forproducing orthogonal translational components, e.g., an O-tRNA, an O-RS,and an O-tRN A/O-RS pair that loads an unnatural amino acid in responseto a selector codon. For example, the negative selection marker, thepositive selection marker or both the positive and negative selectionmarkers can include a marker that fluoresces or catalyzes a luminescentreaction in the presence of a suitable reactant. In another embodiment,a product of the marker is detected by fluorescence-activated cellsorting (FACS) or by luminescence. Optionally, the marker includes anaffinity based screening marker. See also, Francisco, J. A., et al.,(1993) Production and fluorescence-activated cell sorting of Escherichiacoli expressing a functional antibody fragment on the external surface.Proc Natl Acad Sci USA. 90:10444-8.

Additional methods for producing a recombinant orthogonal tRNA can befound, e.g., in International Application Publications WO 2002/086075,entitled “METHODS AND COMPOSITIONS FOR THE PRODUCTION OF ORTHOGONAL tRNAAMINOACYL-tRNA SYNTHETASE PAIRS;” WO 2004/094593, entitled “EXPANDINGTHE EUKARYOTIC GENETIC CODE;” and WO 2005/019415, filed Jul. 7, 2004.See also Forster et al., (2003) Programming peptidomimetic synthetasesby translating genetic codes designed de novo PNAS 100(11):6353-6357;and, Feng et al., (2003), Expanding tRNA recognition of a tRNAsynthetase by a single amino acid change, PNAS 100(10): 5676-5681.

Orthogonal Aminoacyl-tRNA Synthetase (O-RS)

An O-RS of the invention preferentially aminoacylates an O-tRNA with anunnatural amino acid, in vitro or in vivo. An O-RS of the invention canbe provided to the translation system, e.g., a cell, by a polypeptidethat includes an O-RS and/or by a polynucleotide that encodes an O-RS ora portion thereof. For example, an example O-RS comprises an amino acidsequence as set forth in the sequence listing and examples herein, or aconservative variation thereof. In another example, an O-RS, or aportion thereof, is encoded by a polynucleotide sequence that encodes anamino acid comprising sequence in the sequence listing or examplesherein, or a complementary polynucleotide sequence thereof. See, e.g.,the tables and examples herein for sequences of exemplary O-RSmolecules. See also, the section entitled “Nucleic Acid and PolypeptideSequence and Variants” herein.

Methods for identifying an orthogonal aminoacyl-tRNA synthetase (O-RS),e.g., an O-RS, for use with an O-tRNA, are also a feature of theinvention. For example, a method includes subjecting to selection, e.g.,positive selection, a population of cells of a first species, where thecells individually comprise: 1) a member of a plurality ofaminoacyl-tRNA synthetases (RSs), (e.g., the plurality of RSs caninclude mutant RSs, RSs derived from a species other than the firstspecies or both mutant RSs and RSs derived from a species other than thefirst species); 2) the orthogonal tRNA (O-tRNA) (e.g., from one or morespecies); and 3) a polynucleotide that encodes an (e.g., positive)selection marker and comprises at least one selector codon. Cells areselected or screened for those that show an enhancement in suppressionefficiency compared to cells lacking or with a reduced amount of themember of the plurality of RSs. Suppression efficiency can be measuredby techniques known in the art and as described herein. Cells having anenhancement in suppression efficiency comprise an active RS thataminoacylates the O-tRNA. A level of aminoacylation (in vitro or invivo) by the active RS of a first set of tRNAs from the first species iscompared to the level of aminoacylation (in vitro or in vivo) by theactive RS of a second set of tRNAs from the second species. The level ofaminoacylation can be determined by a detectable substance (e.g., alabeled unnatural amino acid). The active RS that more efficientlyaminoacylates the second set of tRNAs compared to the first set of tRNAsis typically selected, thereby providing an efficient (optimized)orthogonal aminoacyl-tRNA synthetase for use with the O-tRNA. An O-RS,identified by the method, is also a feature of the invention.

Any of a number of assays can be used to determine aminoacylation. Theseassays can be performed in vitro or in vivo. For example, in vitroaminoacylation assays are described in, e.g., Hoben and Soil (1985)Methods Enzymol. 113:55-59. Aminoacylation can also be determined byusing a reporter along with orthogonal translation components anddetecting the reporter in a cell expressing a polynucleotide comprisingat least one selector codon that encodes a protein. See also, WO2002/085923, entitled “IN VIVO INCORPORATION OF UNNATURAL AMINO ACIDS;”and WO 2004/094593, entitiled “EXPANDING THE EUKARYOTIC GENETIC CODE.”

Identified O-RS can be further manipulated to alter substratespecificity of the synthetase, so that only a desired unnatural aminoacid, but not any of the common 20 amino acids, are charged to theO-tRNA. Methods to generate an orthogonal aminoacyl tRNA synthetase witha substrate specificity for an unnatural amino acid include mutating thesynthetase, e.g., at the active site in the synthetase, at the editingmechanism site in the synthetase, at different sites by combiningdifferent domains of synthetases, or the like, and applying a selectionprocess. A strategy is used, which is based on the combination of apositive selection followed by a negative selection. In the positiveselection, suppression of the selector codon introduced at anonessential position(s) of a positive marker allows cells to surviveunder positive selection pressure. In the presence of both natural andunnatural amino acids, survivors thus encode active synthetases chargingthe orthogonal suppressor tRNA with either a natural or unnatural aminoacid. In the negative selection, suppression of a selector codonintroduced at a nonessential position(s) of a negative marker removessynthetases with natural amino acid specificities. Survivors of thenegative and positive selection encode synthetases that aminoacylate(charge) the orthogonal suppressor tRNA with unnatural amino acids only.These synthetases can then be subjected to further mutagenesis, e.g.,DNA shuffling or other recursive mutagenesis methods.

A library of mutant O-RSs can be generated using various mutagenesistechniques known in the art. For example, the mutant RSs can begenerated by site-specific mutations, random point mutations, homologousrecombination, DNA shuffling or other recursive mutagenesis methods,chimeric construction or any combination thereof. For example, a libraryof mutant RSs can be produced from two or more other, e.g., smaller,less diverse “sub-libraries.” Chimeric libraries of RSs are alsoincluded in the invention. It should be noted that libraries of tRNAsynthetases from various organism (e.g., microorganisms such aseubacteria or archaebacteria) such as libraries that comprise naturaldiversity (see, e.g., U.S. Pat. No. 6,238,884 to Short et al; U.S. Pat.No. 5,756,316 to Schallenberger et al; U.S. Pat. No. 5,783,431 toPetersen et al; U.S. Pat. No. 5,824,485 to Thompson et al; U.S. Pat. No.5,958,672 to Short et al), are optionally constructed and screened fororthogonal pairs.

Once the synthetases are subject to the positive and negativeselection/screening strategy, these synthetases can then be subjected tofurther mutagenesis. For example, a nucleic acid that encodes the O-RScan be isolated; a set of polynucleotides that encode mutated O-RSs(e.g., by random mutagenesis, site-specific mutagenesis, recombinationor any combination thereof) can be generated from the nucleic acid; and,these individual steps or a combination of these steps can be repeateduntil a mutated O-RS is obtained that preferentially aminoacylates theO-tRNA with the unnatural amino acid. In some aspects of the invention,the steps are performed multiple times, e.g., at least two times.

Additional levels of selection/screening stringency can also be used inthe methods of the invention, for producing O-tRNA, O-RS, or pairsthereof. The selection or screening stringency can be varied on one orboth steps of the method to produce an O-RS. This could include, e.g.,varying the amount of selection/screening agent that is used, etc.Additional rounds of positive and/or negative selections can also beperformed. Selecting or screening can also comprise one or more of achange in amino acid permeability, a change in translation efficiency, achange in translational fidelity, etc. Typically, the one or more changeis based upon a mutation in one or more gene in an organism in which anorthogonal tRNA-tRNA synthetase pair is used to produce protein.

Additional general details for producing O-RS, and altering thesubstrate specificity of the synthetase can be found in InternalPublication Number WO 2002/086075, entitled “METHODS AND COMPOSITIONSFOR THE PRODUCTION OF ORTHOGONAL tRNA AMINOACYL-tRNA SYNTHETASE PAIRS;”and WO 2004/094593, entitled “EXPANDING THE EUKARYOTIC GENETIC CODE.”See also, Wang and Schultz “Expanding the Genetic Code,” AngewandteChemie Int. Ed., 44(1):34-66 (2005), the content of which isincorporated by reference in its entirety.

Source and Host Organisms

The orthogonal translational components (O-tRNA and O-RS) of theinvention can be derived from any organism (or a combination oforganisms) for use in a host translation system from any other species,with the caveat that the O-tRNA/O-RS components and the host system workin an orthogonal manner. It is not a requirement that the O-tRNA and theO-RS from an orthogonal pair be derived from the same organism. In someaspects, the orthogonal components are derived from Archaea genes (i.e.,archaebacteria) for use in a eubacterial host system.

For example, the orthogonal O-tRNA can be derived from an Archaeorganism, e.g., an archaebacterium, such as Methanococcus jannaschii,Methanobacterium thermoautotrophicum, Halobacterium such as Haloferaxvolcanii and Halobacterium species NRC-1, Archaeoglobus fulgidus,Pyrococcus furiosus, Pyrococcus horikoshii, Aeuropyrum pernix,Methanococcus maripaludis, Methanopyrus kandleri, Methanosarcina mazei(Mm), Pyrobaculum aerophilum, Pyrococcus abyssi, Sulfolobus solfataricus(Ss), Sulfolobus tokodaii, Thermoplasma acidophilum, Thermoplasmavolcanium, or the like, or a eubacterium, such as Escherichia coli,Thermus thermophilus, Bacillus stearothermphilus, or the like, while theorthogonal O-RS can be derived from an organism or combination oforganisms, e.g., an archaebacterium, such as Methanococcus jannaschii,Methanobacterium thermoautotrophicum, Halobacterium such as Haloferaxvolcanii and Halobacterium species NRC-1, Archaeoglobus fulgidus,Pyrococcus furiosus, Pyrococcus horikoshii, Aeuropyrum pernix,Methanococcus maripaludis, Methanopyrus kandleri, Methanosarcina mazei,Pyrobaculum aerophilum, Pyrococcus abyssi, Sulfolobus solfataricus,Sulfolobus tokodaii, Thermoplasma acidophilum, Thermoplasma volcanium,or the like, or a eubacterium, such as Escherichia coli, Thermusthermophilus, BacilluS stearothermphilus, or the like. In oneembodiment, eukaryotic sources, e.g., plants, algae, protists, fungi,yeasts, animals (e.g., mammals, insects, arthropods, etc.), or the like,can also be used as sources of O-tRNAs and O-RSs.

The individual components of an O-tRNA/O-RS pair can be derived from thesame organism or different organisms. In one embodiment, the O-tRNA/O-RSpair is from the same organism. Alternatively, the O-tRNA and the O-RSof the O-tRNA/O-RS pair are from different organisms.

The O-tRNA, O-RS or O-tRNA/O-RS pair can be selected or screened in vivoor in vitro and/or used in a cell, e.g., a eubacterial cell, to producea polypeptide with an unnatural amino acid. The eubacterial cell used isnot limited, for example, Escherichia coli, Thermus themzophilus,Bacillus stearothermphilus, or the like. Compositions of eubacterialcells comprising translational components of the invention are also afeature of the invention.

See also, International Application Publication Number WO 2004/094593,entitled “EXPANDING THE EUKARYOTIC GENETIC CODE,” filed Apr. 16, 2004,for screening O-tRNA and/or O-RS in one species for use in anotherspecies.

In some aspects, the O-tRNA, O-RS or O-tRNA/O-RS pair can be selected orscreened in vivo or in vitro and/or used in a cell, e.g., a eukaryoticcell, to produce a polypeptide with an unnatural amino acid. Theeukaryotic cell used is not limited; for example, any suitable yeastcell, such as Saccharomyces cerevisiae (S. cerevisiae) or the like, canbe used. Compositions of eukaryotic cells comprising translationalcomponents of the invention are also a feature of the invention.

Saccharomyces cerevisiae can be chosen as a eukaryotic host species, asthis organism provides various advantages. The species is unicellular,has a rapid generation time, and genetically well characterized. See,e.g., D. Burke, et al., (2000) Methods in Yeast Genetics. Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y. Moreover, since thetranslational machinery of eukaryotes is highly conserved (see, e.g.,(1996) Translational Control. Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y.; Y. Kwok, & J. T. Wong, (1980), Evolutionary relationshipbetween Halobacterium cutirubrum and eukaryotes determined by use ofaminoacyl-tRNA synthetases as phylogenetic probes, Canadian Journal ofBiochemistry 58:213-218; and, (2001) The Ribosome. Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.), O-RS genes (e.g., O-RSgenes derived from wild-type E. coli RS sequences) for the incorporationof unnatural amino acids discovered in S. cerevisiae can be introducedinto higher eukaryotic organisms (e.g., in mammalian cells) and used, inpartnership with cognate tRNAs (see, e.g., K. Sakamoto, et al., (2002)Site-specific incorporation of an unnatural amino acid into proteins inmammalian cells, Nucleic Acids Res. 30:4692-4699; and, C. Kohrer, etal., (2001), Import of amber and ochre suppressor tRNAs into mammaliancells: a general approach to site-specific insertion of amino acidanalogues into proteins, Proc. Natl. Acad. Sci. U.S.A. 98:14310-14315)to incorporate unnatural amino acids.

Although orthogonal translation systems (e.g., comprising an O-RS, anO-tRNA and an unnatural amino acid) can utilize cultured host cells toproduce proteins having unnatural amino acids, it is not intended thatan orthogonal translation system of the invention require an intact,viable host cell. For example, a orthogonal translation system canutilize a cell-free system in the presence of a cell extract. Indeed,the use of cell free, in vitro transcription/translation systems forprotein production is a well established technique. Adaptation of thesein vitro systems to produce proteins having unnatural amino acids usingorthogonal translation system components described herein is well withinthe scope of the invention.

Selector Codons

Selector codons of the invention expand the genetic codon framework ofprotein biosynthetic machinery. For example, a selector codon includes,e.g., a unique three base codon, a nonsense codon, such as a stop codon,e.g., an amber codon (UAG), or an opal codon (UGA), an unnatural codon,at least a four base codon, a rare codon, or the like. A number ofselector codons can be introduced into a desired gene, e.g., one ormore, two or more, more than three, etc. By using different selectorcodons, multiple orthogonal tRNA/synthetase pairs can be used that allowthe simultaneous site-specific incorporation of multiple unnatural aminoacids e.g., including at least one unnatural amino acid, using thesedifferent selector codons.

In one embodiment, the methods involve the use of a selector codon thatis a stop codon for the incorporation of an unnatural amino acid in vivoin a cell. For example, an O-tRNA is produced that recognizes the stopcodon and is aminoacylated by an O-RS with an unnatural amino acid. ThisO-tRNA is not recognized by the naturally occurring host'saminoacyl-tRNA synthetases. Conventional site-directed mutagenesis canbe used to introduce the stop codon at the site of interest in apolynucleotide encoding a polypeptide of interest. See, e.g., Sayers, J.R., et al. (1988), 5′,3′ Exonuclease in phosphorothioate-basedoligonucleotide-directed mutagenesis. Nucleic Acids Res, 791-802. Whenthe O-RS, O-tRNA and the nucleic acid that encodes a polypeptide ofinterest are combined, e.g., in vivo, the unnatural amino acid isincorporated in response to the stop codon to give a polypeptidecontaining the unnatural amino acid at the specified position. In oneembodiment of the invention, the stop codon used as a selector codon isan amber codon, UAG, and/or an opal codon, UGA. In one example, agenetic code in which UAG and UGA are both used as a selector codon canencode 22 amino acids while preserving the ochre nonsense codon, UAA,which is the most abundant termination signal.

The incorporation of unnatural amino acids in vivo can be done withoutsignificant perturbation of the host cell. For example in non-eukaryoticcells, such as Escherichia coli, because the suppression efficiency forthe UAG codon depends upon the competition between the O-tRNA, e.g., theamber suppressor tRNA, and the release factor 1 (RF1) (which binds tothe UAG codon and initiates release of the growing peptide from theribosome), the suppression efficiency can be modulated by, e.g., eitherincreasing the expression level of O-tRNA, e.g., the suppressor tRNA, orusing an RF1 deficient strain. In eukaryotic cells, because thesuppression efficiency for the UAG codon depends upon the competitionbetween the O-tRNA, e.g., the amber suppressor tRNA, and a eukaryoticrelease factor (e.g., eRF) (which binds to a stop codon and initiatesrelease of the growing peptide from the ribosome), the suppressionefficiency can be modulated by, e.g., increasing the expression level ofO-tRNA, e.g., the suppressor tRNA. In addition, additional compounds canalso be present, e.g., reducing agents such as dithiothretiol (DTT).

Unnatural amino acids can also be encoded with rare codons. For example,when the arginine concentration in an in vitro protein synthesisreaction is reduced, the rare arginine codon, AGG, has proven to beefficient for insertion of Ala by a synthetic tRNA acylated withalanine. See, e.g., Ma et al., Biochemistry, 32:7939 (1993). In thiscase, the synthetic tRNA competes with the naturally occurringtRNA^(Arg), which exists as a minor species in Escherichia coli. Inaddition, some organisms do not use all triplet codons. An unassignedcodon AGA in Micrococcus luteus has been utilized for insertion of aminoacids in an in vitro transcription/translation extract. See, e.g., Kowaland Oliver, Nucl. Acid. Res., 25:4685 (1997). Components of theinvention can be generated to use these rare codons in vivo.

Selector codons can also comprise extended codons, e.g., four or morebase codons, such as, four, five, six or more base codons. Examples offour base codons include, e.g., AGGA, CUAG, UAGA, CCCU, and the like.Examples of five base codons include, e.g., AGGAC, CCCCU, CCCUC, CUAGA,CUACU, UAGGC and the like. Methods of the invention include usingextended codons based on frameshift suppression. Four or more basecodons can insert, e.g., one or multiple unnatural amino acids, into thesame protein. In other embodiments, the anticodon loops can decode,e.g., at least a four-base codon, at least a five-base codon, or atleast a six-base codon or more. Since there are 256 possible four-basecodons, multiple unnatural amino acids can be encoded in the same cellusing a four or more base codon. See also, Anderson et al., (2002)Exploring the Limits of Codon and Anticodon Size, Chemistry and Biology,9:237-244; and, Magliery, (2001) Expanding the Genetic Code: Selectionof Efficient Suppressors of Four-base Codons and Identification of“Shifty” Four-base Codons with a Library Approach in Escherichia coli,J. Mol. Biol. 307: 755-769.

For example, four-base codons have been used to incorporate unnaturalamino acids into proteins using in vitro biosynthetic methods. See,e.g., Ma et al., (1993) Biochemistry, 32:7939; and Hohsaka et al.,(1999) J. Am. Chem. Soc., 121:34. CGGG and AGGU were used tosimultaneously incorporate 2-naphthylalanine and an NBD derivative oflysine into streptavidin in vitro with two chemically acylatedframeshift suppressor tRNAs. See, e.g., Hohsaka et al., (1999) J. Am.Chem. Soc., 121:12194. In an in vivo study, Moore et al. examined theability of tRN^(Leu) derivatives with NCUA anticodons to suppress UAGNcodons (N can be U, A, G, or C), and found that the quadruplet UAGA canbe decoded by a tRNA^(Leu) with a UCUA anticodon with an efficiency of13 to 26% with little decoding in the 0 or −1 frame. See Moore et al.,(2000) J. Mol. Biol., 298:195. In one embodiment, extended codons basedon rare codons or nonsense codons can be used in invention, which canreduce missense readthrough and frameshift suppression at other unwantedsites. Four base codons have been used as selector codons in a varietyof orthogonal systems. See, e.g., WO 2005/019415; WO 2005/007870 and WO2005/07624. See also, Wang and Schultz “Expanding the Genetic Code,”Angewandte Chemie int. Ed., 44(1):34-66 (2005), the content of which isincorporated by reference in its entirety. While the examples belowutilize an amber selector codon, four or more base codons can be used aswell, by modifying the examples herein to include four-base O-tRNAs andsynthetases modified to include mutations similar to those previouslydescribed for various unnatural amino acid O-RSs.

For a given system, a selector codon can also include one of the naturalthree base codons, where the endogenous system does not use (or rarelyuses) the natural base codon. For example, this includes a system thatis lacking a tRNA that recognizes the natural three base codon, and/or asystem where the three base codon is a rare codon.

Selector codons optionally include unnatural base pairs. These unnaturalbase pairs further expand the existing genetic alphabet. One extra basepair increases the number of triplet codons from 64 to 125. Propertiesof third base pairs include stable and selective base pairing, efficientenzymatic incorporation into DNA with high fidelity by a polymerase, andthe efficient continued primer extension after synthesis of the nascentunnatural base pair. Descriptions of unnatural base pairs which can beadapted for methods and compositions include, e.g., Hirao, et al.,(2002) An unnatural base pair for incorporating amino acid analoguesinto protein, Nature Biotechnology, 20:177-182. See also Wu, Y., et al.,(2002) J. Am. Chem. Soc. 124:14626-14630. Other relevant publicationsare listed below.

For in vivo usage, the unnatural nucleoside is membrane permeable and isphosphorylated to form the corresponding triphosphate. In addition, theincreased genetic information is stable and not destroyed by cellularenzymes. Previous efforts by Benner and others took advantage ofhydrogen bonding patterns that are different from those in canonicalWatson-Crick pairs, the most noteworthy example of which is theiso-C:iso-G pair. See, e.g., Switzer et al., (1989) J. Am. Chem. Soc.,111:8322; and Piccirilli et al., (1990) Nature, 343:33; Kool, (2000)Curr. Opin. Chem. Biol., 4:602. These bases in general mispair to somedegree with natural bases and cannot be enzymatically replicated. Kooland co-workers demonstrated that hydrophobic packing interactionsbetween bases can replace hydrogen bonding to drive the formation ofbase pair. See Kool, (2000) Curr. Opin. Chem. Biol., 4:602; and Guckianand Kool, (1998) Angew. Chem. Int. Ed. Engl., 36, 2825. In an effort todevelop an unnatural base pair satisfying all the above requirements,Schultz, Romesberg and co-workers have systematically synthesized andstudied a series of unnatural hydrophobic bases. A PICS:PICS self-pairis found to be more stable than natural base pairs, and can beefficiently incorporated into DNA by Klenow fragment of Escherichia coliDNA polymerase I (KF). See, e.g., McMinn et al., (1999) J. Am. Chem.Soc., 121:11586; and Ogawa et al., (2000) J. Am. Chem. Soc., 122:3274. A3MN:3MN self-pair can be synthesized by KF with efficiency andselectivity sufficient for biological function. See, e.g., Ogawa et al.,(2000) J. Am. Chem. Soc., 122:8803. However, both bases act as a chainterminator for further replication. A mutant DNA polymerase has beenrecently evolved that can be used to replicate the PICS self pair. Inaddition, a 7AI self pair can be replicated. See, e.g., Tae et al.,(2001) J. Am. Chem. Soc., 123:7439. A novel metallobase pair, Dipic:Py,has also been developed, which forms a stable pair upon binding Cu(II).See Meggers et al., (2000) J. Am. Chem. Soc., 122:10714. Becauseextended codons and unnatural codons are intrinsically orthogonal tonatural codons, the methods of the invention can take advantage of thisproperty to generate orthogonal tRNAs for them.

A translational bypassing system can also be used to incorporate anunnatural amino acid in a desired polypeptide. In a translationalbypassing system, a large sequence is inserted into a gene but is nottranslated into protein. The sequence contains a structure that servesas a cue to induce the ribosome to hop over the sequence and resumetranslation downstream of the insertion.

Unnatural Amino Acids

As used herein, an unnatural amino acid refers to any amino acid,modified amino acid, or amino acid analogue other than selenocysteineand/or pyrrolysine and the following twenty genetically encodedalpha-amino acids: alanine, arginine, asparagine, aspartic acid,cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine,leucine, lysine, methionine, phenylalanine, proline, serine, threonine,tryptophan, tyrosine, valine. The generic structure of an alpha-aminoacid is illustrated by Formula I:

An unnatural amino acid is typically any structure having Formula Iwherein the R group is any substituent other than one used in the twentynatural amino acids. See e.g., Biochemistry by L. Stryer, 3^(rd) ed.1988, Freeman and Company, New York, for structures of the twentynatural amino acids. Note that, the unnatural amino acids of theinvention can be naturally occurring compounds other than the twentyalpha-amino acids above.

Because the unnatural amino acids of the invention typically differ fromthe natural amino acids in side chain, the unnatural amino acids formamide bonds with other amino acids, e.g., natural or unnatural, in thesame manner in which they are formed in naturally occurring proteins.However, the unnatural amino acids have side chain groups thatdistinguish them from the natural amino acids.

Of particular interest herein are unnatural amino acids provided inFIG. 1. For example, these unnatural amino acids include but are notlimited to p-ethylthiocarbonyl-L-phenylalanine,p-(3-oxobutanoyl)-L-phenylalanine, 1,5-dansyl-alanine, 7-amino-coumarinamino acid, 7-hydroxy-coumarin amino acid, nitrobenzyl-serine,O-(2-nitrobenzyl)-L-tyrosine, p-carboxymethyl-L-phenylalanine,p-cyano-L-phenylalanine, m-cyano-L-phenylalanine, biphenylalanine,3-amino-L-tyrosine, bipyridyl alanine,p-(2-amino-1-hydroxyethyl)-L-phenylalanine,p-isopropylthiocarbonyl-L-phenylalanine, 3-nitro-L-tyrosine andp-nitro-L-phenylalanine. Both the L and D-enantiomers of these unnaturalamino acids find use with the invention

In addition to the unnatural amino acids of FIG. 1, other unnaturalamino acids can be simultaneously incorporated into a polypeptide ofinterest, e.g., using an appropriate second O-RS/O-tRNA pair inconjunction with an orthogonal pair provided by the present invention.Many such additional unnatural amino acids and suitable orthogonal pairsare known. See the references cited herein. For example, see Wang andSchultz “Expanding the Genetic Code,” Angewandte Chemie Int. Ed.,44(1):34-66 (2005), the content of which is incorporated by reference inits entirety.

In other unnatural amino acids, for example, R in Formula I optionallycomprises an alkyl-, aryl-, acyl-, hydrazine, cyano-, halo-, hydrazide,alkenyl, ether, borate, boronate, phospho, phosphono, phosphine, enone,imine, ester, hydroxylamine, amine, and the like, or any combinationthereof. Other unnatural amino acids of interest include, but are notlimited to, amino acids comprising a photoactivatable cross-linker,spin-labeled amino acids, fluorescent amino acids, metal binding aminoacids, metal-containing amino acids, radioactive amino acids, aminoacids with novel functional groups, amino acids that covalently ornoncovalently interact with other molecules, photocaged and/orphotoisomerizable amino acids, biotin or biotin-analogue containingamino acids, keto containing amino acids, glycosylated amino acids, asaccharide moiety attached to the amino acid side chain, amino acidscomprising polyethylene glycol or polyether, heavy atom substitutedamino acids, chemically cleavable or photocleavable amino acids, aminoacids with an elongated side chain as compared to natural amino acids(e.g., polyethers or long chain hydrocarbons, e.g., greater than about5, greater than about 10 carbons, etc.), carbon-linked sugar-containingamino acids, amino thioacid containing amino acids, and amino acidscontaining one or more toxic moiety.

In another aspect, the invention provides unnatural amino acids havingthe general structure illustrated by Formula IV below:

An unnatural amino acid having this structure is typically any structurewhere R₁ is a substituent used in one of the twenty natural amino acids(e.g., tyrosine or phenylalanine) and R₂ is a substituent. Thus, thistype of unnatural amino acid can be viewed as a natural amino acidderivative.

In addition to unnatural amino acids that contain novel side chains suchas those shown in FIG. 1, unnatural amino acids can also optionallycomprise modified backbone structures, e.g., as illustrated by thestructures of Formula II and III:

wherein Z typically comprises OH, NH₂, SH, NH—R′, or S—R′; X and Y,which can be the same or different, typically comprise S or O, and R andR′, which are optionally the same or different, are typically selectedfrom the same list of constituents for the R group described above forthe unnatural amino acids having Formula I as well as hydrogen. Forexample, unnatural amino acids of the invention optionally comprisesubstitutions in the amino or carboxyl group as illustrated by FormulasII and III. Unnatural amino acids of this type include, but are notlimited to, α-hydroxy acids, α-thioacids α-aminothiocarboxylates, e.g.,with side chains corresponding to the common twenty natural amino acidsor unnatural side chains. In addition, substitutions at the α-carbonoptionally include L, D, or α-α-disubstituted amino acids such asD-glutamate, D-alanine, D-methyl-O-tyrosine, aminobutyric acid, and thelike. Other structural alternatives include cyclic amino acids, such asproline analogues as well as 3,4,6,7,8, and 9 membered ring prolineanalogues, β and γ amino acids such as substituted β-alanine and γ-aminobutyric acid.

In some aspects, the invention utilizes unnatural amino acids in theL-configuration. However, it is not intended that the invention belimited to the use of L-configuration unnatural amino acids. It iscontemplated that the D-enantiomers of these unnatural amino acids alsofind use with the invention.

Tyrosine analogs include para-substituted tyrosines, ortho-substitutedtyrosines, and meta substituted tyrosines, wherein the substitutedtyrosine comprises an alkynyl group, acetyl group, a benzoyl group, anamino group, a hydrazine, an hydroxyamine, a thiol group, a carboxygroup, an isopropyl group, a methyl group, a C₆-C₂₀ straight chain orbranched hydrocarbon, a saturated or unsaturated hydrocarbon, anO-methyl group, a polyether group, a nitro group, or the like. Inaddition, multiply substituted aryl rings are also contemplated.Glutamine analogs of the invention include, but are not limited to,α-hydroxy derivatives, γ-substituted derivatives, cyclic derivatives,and amide substituted glutamine derivatives. Example phenylalanineanalogs include, but are not limited to, para-substitutedphenylalanines, ortho-substituted phenyalanines, and meta-substitutedphenylalanines, wherein the substituent comprises an alkynyl group, ahydroxy group, a methoxy group, a methyl group, an allyl group, analdehyde, a nitro, a thiol group, or keto group, or the like. Specificexamples of unnatural amino acids include, but are not limited to,p-ethylthiocarbonyl-L-phenylalanine, p-(3-oxobutanoyl)-L-phenylalanine,1,5-dansyl-alanine, 7-amino-coumarin amino acid, 7-hydroxy-coumarinamino acid, nitrobenzyl-serine, O-(2-nitrobenzyl)-L-tyrosine,p-carboxymethyl-L-phenylalanine, p-cyano-L-phenylalanine,m-cyano-L-phenylalanine, biphenylalanine, 3-amino-L-tyrosine, bipyridylalanine, p-(2-amino-1-hydroxyethyl)-L-phenylalanine,p-isopropylthiocarbonyl-L-phenylalanine, 3-nitro-L-tyrosine andp-nitro-L-phenylalanine. Also, a p-propargyloxyphenylalanine, a3,4-dihydroxy-L-phenyalanine (DHP), a 3,4,6-trihydroxy-L-phenylalanine,a 3,4,5-trihydroxy-L-phenylalanine, 4-nitro-phenylalanine, ap-acetyl-L-phenylalanine, O-methyl-L-tyrosine, anL-3-(2-naphthyl)alanine, a 3-methyl-phenylalanine, anO-4-allyl-L-tyrosine, a 4-propyl-L-tyrosine, a 3-nitro-tyrosine, a3-thiol-tyrosine, a tri-O-acetyl-GlcNAcβ-serine, an L-Dopa, afluorinated phenylalanine, an isopropyl-L-phenylalanine, ap-azido-L-phenylalanine, a p-acyl-L-phenylalanine, ap-benzoyl-L-phenylalanine, an L-phosphoserine, a phosphonoserine, aphosphonotyrosine, a p-iodo-phenylalanine, a p-bromophenylalanine, ap-amino-L-phenylalanine, and an isopropyl-L-phenylalanine, and the like.The structures of a variety of unnatural amino acids are providedherein, see, for example, FIG. 1. See also, Published InternationalApplication WO 2004/094593, entitled “EXPANDING THE EUKARYOTIC GENETICCODE.”

Chemical Synthesis of Unnatural Amino Acids

Many of the unnatural amino acids provided above are commerciallyavailable, e.g., from Sigma (USA) or Aldrich (Milwaukee, Wis., USA).Those that are not commercially available are optionally synthesized asprovided in various publications or using standard methods known tothose of skill in the art. For organic synthesis techniques, see, e.g.,Organic Chemistry by Fessendon and Fessendon, (1982, Second Edition,Willard Grant Press, Boston Mass.); Advanced Organic Chemistry by March(Third Edition, 1985, Wiley and Sons, New York); and Advanced OrganicChemistry by Carey and Sundberg (Third Edition, Parts A and B, 1990,Plenum Press, New York). Additional publications describing thesynthesis of unnatural amino acids include, e.g., WO 2002/085923entitled “In vivo incorporation of Unnatural Amino Acids;” Matsoukas etal., (1995) J. Med. Chem., 38, 4660-4669; King, F. E. & Kidd, D. A. A.(1949) A New Synthesis of Glutamine and of γ-Dipeptides of Glutamic Acidfrom Phthylated Intermediates. J. Chem. Soc., 3315-3319; Friedman, O. M.& Chatterrji, R. (1959) Synthesis of Derivatives of Glutamine as ModelSubstrates for Anti-Tumor Agents. J. Am. Chem. Soc. 81, 3750-3752;Craig, J. C. et al. (1988) Absolute Configuration of the Enantiomers of7-Chloro-4 [[4-(diethylamino)-1-methylbutyl]amino]quinoline(Chloroquine). J. Org. Chem. 53, 1167-1170; Azoulay, M., Vilmont, M. &Frappier, F. (1991) Glutamine analogues as Potential Antimalarials.,Eur. J. Med. Chem. 26, 201-5; Koskinen, A. M. P. & Rapoport, H. (1989)Synthesis of 4-Substituted Prolines as Conformationally ConstrainedAmino Acid Analogues. J. Org. Chem. 54, 1859-1866; Christie, B. D. &Rapoport, H. (1985) Synthesis of Optically Pure Pipecolates fromL-Asparagine. Application to the Total Synthesis of (+)-Apovincaminethrough Amino Acid Decarbonylation and Iminium Ion Cyclization. J. Org.Chem. 1989:1859-1866; Barton et al., (1987) Synthesis of Novela-Amino-Acids and Derivatives Using Radical Chemistry: Synthesis ofL-and D-a-Amino-Adipic Acids, L-a-aminopimelic Acid and AppropriateUnsaturated Derivatives. Tetrahedron Lett. 43:4297-4308; and, Subasingheet al., (1992) Quisqualic acid analogues: synthesis of beta-heterocyclic2-aminopropanoic acid derivatives and their activity at a novelquisqualate-sensitized site. J. Med. Chem. 35:4602-7. See also,International Publication WO 2004/058946, entitled “PROTEIN ARRAYS,”filed on Dec. 22, 2003.

Cellular Uptake of Unnatural Amino Acids

Unnatural amino acid uptake by a cell is one issue that is typicallyconsidered when designing and selecting unnatural amino acids, e.g., forincorporation into a protein. For example, the high charge density ofα-amino acids suggests that these compounds are unlikely to be cellpermeable. Natural amino acids are taken up into the cell via acollection of protein-based transport systems often displaying varyingdegrees of amino acid specificity. A rapid screen can be done whichassesses which unnatural amino acids, if any, are taken up by cells.See, e.g., the toxicity assays in, e.g., International Publication WO2004/058946, entitled “PROTEIN ARRAYS,” filed on Dec. 22, 2003; and Liuand Schultz (1999) Progress toward the evolution of an organism with anexpanded genetic code. PNAS 96:4780-4785. Although uptake is easilyanalyzed with various assays, an alternative to designing unnaturalamino acids that are amenable to cellular uptake pathways is to providebiosynthetic pathways to create amino acids in vivo.

Biosynthesis of Unnatural Amino Acids

Many biosynthetic pathways already exist in cells for the production ofamino acids and other compounds. While a biosynthetic method for aparticular unnatural amino acid may not exist in nature, e.g., in acell, the invention provides such methods. For example, biosyntheticpathways for unnatural amino acids are optionally generated in host cellby adding new enzymes or modifying existing host cell pathways.Additional new enzymes are optionally naturally occurring enzymes orartificially evolved enzymes. For example, the biosynthesis ofp-aminophenylalanine (as presented in an example in WO 2002/085923,supra) relies on the addition of a combination of known enzymes fromother organisms. The genes for these enzymes can be introduced into acell by transforming the cell with a plasmid comprising the genes. Thegenes, when expressed in the cell, provide an enzymatic pathway tosynthesize the desired compound. Examples of the types of enzymes thatare optionally added are provided in the examples below. Additionalenzymes sequences are found, e.g., in Genbank. Artificially evolvedenzymes are also optionally added into a cell in the same manner. Inthis manner, the cellular machinery and resources of a cell aremanipulated to produce unnatural amino acids.

Indeed, any of a variety of methods can be used for producing novelenzymes for use in biosynthetic pathways, or for evolution of existingpathways, for the production of unnatural amino acids, in vitro or invivo. Many available methods of evolving enzymes and other biosyntheticpathway components can be applied to the present invention to produceunnatural amino acids (or, indeed, to evolve synthetases to have newsubstrate specificities or other activities of interest). For example,DNA shuffling is optionally used to develop novel enzymes and/orpathways of such enzymes for the production of unnatural amino acids (orproduction of new synthetases), in vitro or in vivo. See, e.g., Stemmer(1994), Rapid evolution of a protein in vitro by DNA shuffling, Nature370(4):389-391; and, Stemmer, (1994), DNA shuffling by randomfragmentation and reassembly: In vitro recombination for molecularevolution, Proc. Natl. Acad. Sci. USA., 91:10747-10751. A relatedapproach shuffles families of related (e.g., homologous) genes toquickly evolve enzymes with desired characteristics. An example of such“family gene shuffling” methods is found in Crameri et al. (1998) “DNAshuffling of a family of genes from diverse species accelerates directedevolution” Nature, 391(6664): 288-291. New enzymes (whether biosyntheticpathway components or synthetases) can also be generated using a DNArecombination procedure known as “incremental truncation for thecreation of hybrid enzymes” (“ITCHY”), e.g., as described in Ostermeieret al. (1999) “A combinatorial approach to hybrid enzymes independent ofDNA homology” Nature Biotech 17:1205. This approach can also be used togenerate a library of enzyme or other pathway variants which can serveas substrates for one or more in vitro or in vivo recombination methods.See, also, Ostermeier et al. (1999) “Combinatorial Protein Engineeringby Incremental Truncation,” Proc. Natl. Acad. Sci. USA, 96: 3562-67, andOstermeier et al. (1999), “Incremental Truncation as a Strategy in theEngineering of Novel Biocatalysts,” Biological and Medicinal Chemistry,7: 2139-44. Another approach uses exponential ensemble mutagenesis toproduce libraries of enzyme or other pathway variants that are, e.g.,selected for an ability to catalyze a biosynthetic reaction relevant toproducing an unnatural amino acid (or a new synthetase). In thisapproach, small groups of residues in a sequence of interest arerandomized in parallel to identify, at each altered position, aminoacids which lead to functional proteins. Examples of such procedures,which can be adapted to the present invention to produce new enzymes forthe production of unnatural amino acids (or new synthetases) are foundin Delegrave & Youvan (1993) Biotechnology Research 11:1548-1552. In yetanother approach, random or semi-random mutagenesis using doped ordegenerate oligonucleotides for enzyme and/or pathway componentengineering can be used, e.g., by using the general mutagenesis methodsof e.g., Arkin and Youvan (1992) “Optimizing nucleotide mixtures toencode specific subsets of amino acids for semi-random mutagenesis”Biotechnology 10:297-300; or Reidhaar-Olson et al. (1991) “Randommutagenesis of protein sequences using oligonucleotide cassettes”Methods Enzymol. 208:564-86. Yet another approach, often termed a“non-stochastic” mutagenesis, which uses polynucleotide reassembly andsite-saturation mutagenesis can be used to produce enzymes and/orpathway components, which can then be screened for an ability to performone or more synthetase or biosynthetic pathway function (e.g., for theproduction of unnatural amino acids in vivo). See, e.g., Short“NON-STOCHASTIC GENERATION OF GENETIC VACCINES AND ENZYMES” WO 00/46344.

An alternative to such mutational methods involves recombining entiregenomes of organisms and selecting resulting progeny for particularpathway functions (often referred to as “whole genome shuffling”). Thisapproach can be applied to the present invention, e.g., by genomicrecombination and selection of an organism (e.g., an E. coli or othercell) for an ability to produce an unnatural amino acid (or intermediatethereof). For example, methods taught in the following publications canbe applied to pathway design for the evolution of existing and/or newpathways in cells to produce unnatural amino acids in vivo: Patnaik etal. (2002) “Genome shuffling of lactobacillus for improved acidtolerance” Nature Biotechnology, 20(7): 707-712; and Zhang et al. (2002)“Genome shuffling leads to rapid phenotypic improvement in bacteria”Nature, February 7, 415(6872): 644-646.

Other techniques for organism and metabolic pathway engineering, e.g.,for the production of desired compounds are also available and can alsobe applied to the production of unnatural amino acids. Examples ofpublications teaching useful pathway engineering approaches include:Nakamura and White (2003) “Metabolic engineering for the microbialproduction of 1,3 propanediol” Curr. Opin. Biotechnol. 14(5):454-9;Berry et al. (2002) “Application of Metabolic Engineering to improveboth the production and use of Biotech Indigo” J. IndustrialMicrobiology and Biotechnology 28:127-133; Banta et al. (2002)“Optimizing an artificial metabolic pathway: Engineering the cofactorspecificity of Corynebacterium 2,5-diketo-D-gluconic acid reductase foruse in vitamin C biosynthesis” Biochemistry, 41(20), 6226-36; Selivonovaet al. (2001) “Rapid Evolution of Novel Traits in Microorganisms”Applied and Environmental Microbiology, 67:3645, and many others.

Regardless of the method used, typically, the unnatural amino acidproduced with an engineered biosynthetic pathway of the invention isproduced in a concentration sufficient for efficient proteinbiosynthesis, e.g., a natural cellular amount, but not to such a degreeas to significantly affect the concentration of other cellular aminoacids or to exhaust cellular resources. Typical concentrations producedin vivo in this manner are about 10 mM to about 0.05 mM. Once a cell isengineered to produce enzymes desired for a specific pathway and anunnatural amino acid is generated, in vivo selections are optionallyused to further optimize the production of the unnatural amino acid forboth ribosomal protein synthesis and cell growth.

Orthogonal Components for Incorporating Unnatural Amino Acids

The invention provides compositions and methods of producing orthogonalcomponents for incorporating unnatural amino acids, e.g., the unnaturalamino acids provided in FIG. 1, into a growing polypeptide chain inresponse to a selector codon, e.g., an amber stop codon, a nonsensecodon, a four or more base codon, etc., e.g., in vivo. For example, theinvention provides orthogonal-tRNAs (O-tRNAs), orthogonal aminoacyl-tRNAsynthetases (O-RSs) and pairs thereof. These pairs can be used toincorporate an unnatural amino acid into growing polypeptide chains.

A composition of the invention includes an orthogonal aminoacyl-tRNAsynthetase (O-RS), where the O-RS preferentially aminoacylates an O-tRNAwith p-ethylthiocarbonyl-L-phenylalanine,p-(3-oxobutanoyl)-L-phenylalanine, 1,5-dansyl-alanine, 7-amino-coumarinalanine, 7-hydroxy-coumarin alanine, o-nitrobenzyl-serine,O-(2-nitrobenzyl)-L-tyrosine, p-carboxymethyl-L-phenylalanine,p-cyano-L-phenylalanine, m-cyano-L-phenylalanine, biphenylalanine,3-amino-L-tyrosine, bipyridylalanine,p-(2-amino-1-hydroxyethyl)-L-phenylalanine;p-isopropylthiocarbonyl-L-phenylalanine; 3-nitro-L-tyrosine orp-nitro-L-phenylalanine. In certain embodiments, the O-RS comprises anamino acid sequence comprising any one of SEQ ID NOS: 7-10, 12, 14, 16,18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 50, 52-55,57 and 59-63, and conservative variations thereof. In certainembodiments of the invention, the O-RS preferentially aminoacylates theO-tRNA over any endogenous tRNA with an the particular unnatural aminoacid, where the O-RS has a bias for the O-tRNA, and where the ratio ofO-tRNA charged with an unnatural amino acid to the endogenous tRNAcharged with the same unnatural amino acid is greater than 1:1, and morepreferably where the O-RS charges the O-tRNA exclusively or nearlyexclusively.

A composition that includes an O-RS can optionally further include anorthogonal tRNA (O-tRNA), where the O-tRNA recognizes a selector codon.Typically, an O-tRNA of the invention includes at least about, e.g., a45%, a 50%, a 60%, a 75%, an 80%, or a 90% or more suppressionefficiency in the presence of a cognate synthetase in response to aselector codon as compared to the suppression efficiency of an O-tRNAcomprising or encoded by a polynucleotide sequence as set forth in thesequence listings (e.g., SEQ ID NO: 1) and examples herein. In oneembodiment, the suppression efficiency of the O-RS and the O-tRNAtogether is, e.g., 5 fold, 10 fold, 15 fold, 20 fold, 25 fold or moregreater than the suppression efficiency of the O-tRNA in the absence ofan O-RS. In some aspects, the suppression efficiency of the O-RS and theO-tRNA together is at least 45% of the suppression efficiency of anorthogonal tyrosyl-tRNA synthetase pair derived from Methanococcusjannaschii, or alternatively, an orthogonal leucyl-tRNA synthetase pairderived from E. coli.

A composition that includes an O-tRNA can optionally include a cell(e.g., a eubacterial cell, such as an E. coli cell and the like, or aeukaryotic cell such as a yeast cell), and/or a translation system.

A cell (e.g., a eubacterial cell or a yeast cell) comprising atranslation system is also provided by the invention, where thetranslation system includes an orthogonal-tRNA (O-tRNA); an orthogonalaminoacyl-tRNA synthetase (O-RS); and, an unnatural amino acid, e.g., anamino acid shown in FIG. 1. Typically, the O-RS preferentiallyaminoacylates the O-tRNA over any endogenous tRNA with the unnaturalamino acid, where the O-RS has a bias for the O-tRNA, and where theratio of O-tRNA charged with the unnatural amino acid to the endogenoustRNA charged with the unnatural amino acid is greater than 1:1, and morepreferably where the O-RS charges the O-tRNA exclusively or nearlyexclusively. The O-tRNA recognizes the first selector codon, and theO-RS preferentially aminoacylates the O-tRNA with an unnatural aminoacid. In one embodiment, the O-tRNA comprises or is encoded by apolynucleotide sequence as set forth in SEQ ID NO: 1, SEQ ID NO: 2, or acomplementary polynucleotide sequence thereof. In one embodiment, theO-RS comprises an amino acid sequence as set forth in any one of SEQ IDNOS: 7-10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40,42, 44, 46, 50, 52-55, 57, 59-63, and conservative variations thereof.

A cell of the invention can optionally further comprise an additionaldifferent O-tRNA/O-RS pair and a second unnatural amino acid, e.g.,where this O-tRNA recognizes a second selector codon and this O-RSpreferentially aminoacylates the corresponding O-tRNA with the secondunnatural amino acid, where the second amino acid is different from thefirst unnatural amino acid. Optionally, a cell of the invention includesa nucleic acid that comprises a polynucleotide that encodes apolypeptide of interest, where the polynucleotide comprises a selectorcodon that is recognized by the O-tRNA.

In certain embodiments, a cell of the invention is a eubacterial cell(such as E. coli) or a yeast cell, that includes an orthogonal-tRNA(O-tRNA), an orthogonal aminoacyl-tRNA synthetase (O-RS), an unnaturalamino acid, and a nucleic acid that comprises a polynucleotide thatencodes a polypeptide of interest, where the polynucleotide comprisesthe selector codon that is recognized by the O-tRNA. In certainembodiments of the invention, the O-RS preferentially aminoacylates theO-tRNA with the unnatural amino acid with an efficiency that is greaterthan the efficiency with which the O-RS aminoacylates any endogenoustRNA.

In certain embodiments of the invention, an O-tRNA of the inventioncomprises or is encoded by a polynucleotide sequence as set forth in thesequence listings (e.g., SEQ ID NO: 1 or SEQ ID NO: 2) or examplesherein, or a complementary polynucleotide sequence thereof. In certainembodiments of the invention, an O-RS comprises an amino acid sequenceas set forth in the sequence listings, or a conservative variationthereof. In one embodiment, the O-RS or a portion thereof is encoded bya polynucleotide sequence encoding an amino acid as set forth in thesequence listings or examples herein, or a complementary polynucleotidesequence thereof.

The O-tRNA and/or the O-RS of the invention can be derived from any of avariety of organisms (e.g., eukaryotic and/or non-eukaryotic organisms).

Polynucleotides are also a feature of the invention. A polynucleotide ofthe invention includes an artificial (e.g., man-made, and not naturallyoccurring) polynucleotide comprising a nucleotide sequence encoding apolypeptide as set forth in the sequence listings herein, and/or iscomplementary to or that polynucleotide sequence. A polynucleotide ofthe invention can also includes a nucleic acid that hybridizes to apolynucleotide described above, under highly stringent conditions, oversubstantially the entire length of the nucleic acid. A polynucleotide ofthe invention also includes a polynucleotide that is, e.g., at least75%, at least 80%, at least 90%, at least 95%, at least 98% or moreidentical to that of a naturally occurring tRNA or corresponding codingnucleic acid (but a polynucleotide of the invention is other than anaturally occurring tRNA or corresponding coding nucleic acid), wherethe tRNA recognizes a selector codon, e.g., a four base codon.Artificial polynucleotides that are, e.g., at least 80%, at least 90%,at least 95%, at least 98% or more identical to any of the above and/ora polynucleotide comprising a conservative variation of any the above,are also included in polynucleotides of the invention.

Vectors comprising a polynucleotide of the invention are also a featureof the invention. For example, a vector of the invention can include aplasmid, a cosmid, a phage, a virus, an expression vector, and/or thelike. A cell comprising a vector of the invention is also a feature ofthe invention.

Methods of producing components of an O-tRNA/O-RS pair are also featuresof the invention. Components produced by these methods are also afeature of the invention. For example, methods of producing at least onetRNA that is orthogonal to a cell (O-tRNA) include generating a libraryof mutant tRNAs; mutating an anticodon loop of each member of thelibrary of mutant tRNAs to allow recognition of a selector codon,thereby providing a library of potential O-tRNAs, and subjecting tonegative selection a first population of cells of a first species, wherethe cells comprise a member of the library of potential O-tRNAs. Thenegative selection eliminates cells that comprise a member of thelibrary of potential O-tRNAs that is aminoacylated by an aminoacyl-tRNAsynthetase (RS) that is endogenous to the cell. This provides a pool oftRNAs that are orthogonal to the cell of the first species, therebyproviding at least one O-tRNA. An O-tRNA produced by the methods of theinvention is also provided.

In certain embodiments, the methods further comprise subjecting topositive selection a second population of cells of the first species,where the cells comprise a member of the pool of tRNAs that areorthogonal to the cell of the first species, a cognate aminoacyl-tRNAsynthetase, and a positive selection marker. Using the positiveselection, cells are selected or screened for those cells that comprisea member of the pool of tRNAs that is aminoacylated by the cognateaminoacyl-tRNA synthetase and that shows a desired response in thepresence of the positive selection marker, thereby providing an O-tRNA.In certain embodiments, the second population of cells comprise cellsthat were not eliminated by the negative selection.

Methods for identifying an orthogonal-aminoacyl-tRNA synthetase thatcharges an O-tRNA with an unnatural amino acid are also provided. Forexample, methods include subjecting a population of cells of a firstspecies to a selection, where the cells each comprise: 1) a member of aplurality of aminoacyl-tRNA synthetases (RSs), (e.g., the plurality ofRSs can include mutant RSs, RSs derived from a species other than afirst species or both mutant RSs and RSs derived from a species otherthan a first species); 2) the orthogonal-tRNA (O-tRNA) (e.g., from oneor more species); and 3) a polynucleotide that encodes a positiveselection marker and comprises at least one selector codon.

Cells (e.g., a host cell) are selected or screened for those that showan enhancement in suppression efficiency compared to cells lacking orhaving a reduced amount of the member of the plurality of RSs. Theseselected/screened cells comprise an active RS that aminoacylates theO-tRNA. An orthogonal aminoacyl-tRNA synthetase identified by the methodis also a feature of the invention.

Methods of producing a protein in a cell (e.g., in a eubacterial cellsuch as an E. coli cell or the like, or in a yeast cell) having theunnatural amino acid at a selected position are also a feature of theinvention. For example, a method includes growing, in an appropriatemedium, a cell, where the cell comprises a nucleic acid that comprisesat least one selector codon and encodes a protein, providing theunnatural amino acid, and incorporating the unnatural amino acid intothe specified position in the protein during translation of the nucleicacid with the at least one selector codon, thereby producing theprotein. The cell further comprises: an orthogonal-tRNA (O-tRNA) thatfunctions in the cell and recognizes the selector codon; and, anorthogonal aminoacyl-tRNA synthetase (O-RS) that preferentiallyaminoacylates the O-tRNA with the unnatural amino acid. A proteinproduced by this method is also a feature of the invention.

The invention also provides compositions that include proteins, wherethe proteins comprise, e.g., p-ethylthiocarbonyl-L-phenylalanine,p-(3-oxobutanoyl)-L-phenylalanine, 1,5-dansyl-alanine, 7-amino-coumarinamino acid, 7-hydroxy-coumarin amino acid, nitrobenzyl-serine,O-(2-nitrobenzyl)-L-tyrosine, p-carboxymethyl-L-phenylalanine,p-cyano-L-phenylalanine, m-cyano-L-phenylalanine, biphenylalanine,3-amino-L-tyrosine, bipyridyl alanine,p-(2-amino-1-hydroxyethyl)-L-phenylalanine,p-isopropylthiocarbonyl-L-phenylalanine, 3-nitro-L-tyrosine orp-nitro-L-phenylalanine. In certain embodiments, the protein comprisesan amino acid sequence that is at least 75% identical to that of a knownprotein, e.g., a therapeutic protein, a diagnostic protein, anindustrial enzyme, or portion thereof. Optionally, the compositioncomprises a pharmaceutically acceptable carrier.

Nucleic Acid and Polypeptide Sequence and Variants

As described herein, the invention provides for polynucleotide sequencesencoding, e.g., O-tRNAs and O-RSs, and polypeptide amino acid sequences,e.g., O-RSs, and, e.g., compositions, systems and methods comprisingsaid polynucleotide or polypeptide sequences. Examples of saidsequences, e.g., O-tRNA and O-RS amino acid and nucleotide sequences aredisclosed herein (see Table 5, e.g., SEQ ID NOS: 7-10, 12, 14, 16, 18,20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 50, 52-55, 57and 59-63). However, one of skill in the art will appreciate that theinvention is not limited to those sequences disclosed herein, e.g., inthe Examples and sequence listing. One of skill will appreciate that theinvention also provides many related sequences with the functionsdescribed herein, e.g., polynucleotides and polypeptides encodingconservative variants of an O-RS disclosed herein.

The construction and analysis of orthogonal synthetase species (O-RS)that are able to aminoacylate an O-tRNA with an unnatural amino acid,for example, an unnatural amino acid provided in FIG. 1, are describedin Examples 1 through 16. These Examples describe the construction andanalysis of O-RS species that are able to incorporate the unnaturalamino acids p-ethylthiocarbonyl-L-phenylalanine,p-(3-oxobutanoyl)-L-phenylalanine, 1,5-dansyl-alanine, 7-amino-coumarinalanine, 7-hydroxy-coumarin alanine, o-nitrobenzyl-serine,O-(2-nitrobenzyl)-L-tyrosine, p-carboxymethyl-L-phenylalanine,p-cyano-L-phenylalanine, m-cyano-L-phenylalanine, biphenylalanine,3-amino-L-tyrosine, bipyridylalanine,p-(2-amino-1-hydroxyethyl)-L-phenylalanine;p-isopropylthiocarbonyl-L-phenylalanine; 3-nitro-L-tyrosine andp-nitro-L-phenylalanine.

The invention provides polypeptides (O-RSs) and polynucleotides, e.g.,O-tRNA, polynucleotides that encode O-RSs or portions thereof,oligonucleotides used to isolate aminoacyl-tRNA synthetase clones, etc.Polynucleotides of the invention include those that encode proteins orpolypeptides of interest of the invention with one or more selectorcodon. In addition, polynucleotides of the invention include, e.g., apolynucleotide comprising a nucleotide sequence as set forth in SEQ IDNO: 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43,45, 47, 51, 56 and 58, and a polynucleotide that is complementary to orthat encodes a polynucleotide sequence thereof. A polynucleotide of theinvention also includes any polynucleotide that encodes an O-RS aminoacid sequence comprising SEQ ID NO: 7-10, 12, 14, 16, 18, 20, 22, 24,26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 50, 52-55, 57 and 59-63.Similarly, an artificial nucleic acid that hybridizes to apolynucleotide indicated above under highly stringent conditions oversubstantially the entire length of the nucleic acid (and is other than anaturally occurring polynucleotide) is a polynucleotide of theinvention. In one embodiment, a composition includes a polypeptide ofthe invention and an excipient (e.g., buffer, water, pharmaceuticallyacceptable excipient, etc.). The invention also provides an antibody orantisera specifically immunoreactive with a polypeptide of theinvention. An artificial polynucleotide is a polynucleotide that is manmade and is not naturally occurring.

A polynucleotide of the invention also includes an artificialpolynucleotide that is, e.g., at least 75%, at least 80%, at least 90%,at least 95%, at least 98% or more identical to that of a naturallyoccurring tRNA, (but is other than a naturally occurring tRNA). Apolynucleotide also includes an artificial polynucleotide that is, e.g.,at least 75%, at least 80%, at least 90%, at least 95%, at least 98% ormore identical (but not 100% identical) to that of a naturally occurringtRNA.

In certain embodiments, a vector (e.g., a plasmid, a cosmid, a phage, avirus, etc.) comprises a polynucleotide of the invention. In oneembodiment, the vector is an expression vector. In another embodiment,the expression vector includes a promoter operably linked to one or moreof the polynucleotides of the invention. In another embodiment, a cellcomprises a vector that includes a polynucleotide of the invention.

One of skill will also appreciate that many variants of the disclosedsequences are included in the invention. For example, conservativevariations of the disclosed sequences that yield a functionallyidentical sequence are included in the invention. Variants of thenucleic acid polynucleotide sequences, wherein the variants hybridize toat least one disclosed sequence, are considered to be included in theinvention. Unique subsequences of the sequences disclosed herein, asdetermined by, e.g., standard sequence comparison techniques, are alsoincluded in the invention.

Conservative Variations

Owing to the degeneracy of the genetic code, “silent substitutions”(i.e., substitutions in a nucleic acid sequence which do not result inan alteration in an encoded polypeptide) are an implied feature of everynucleic acid sequence that encodes an amino acid sequence. Similarly,“conservative amino acid substitutions,” where one or a limited numberof amino acids in an amino acid sequence are substituted with differentamino acids with highly similar properties, are also readily identifiedas being highly similar to a disclosed construct. Such conservativevariations of each disclosed sequence are a feature of the presentinvention.

“Conservative variations” of a particular nucleic acid sequence refersto those nucleic acids which encode identical or essentially identicalamino acid sequences, or, where the nucleic acid does not encode anamino acid sequence, to essentially identical sequences. One of skillwill recognize that individual substitutions, deletions or additionswhich alter, add or delete a single amino acid or a small percentage ofamino acids (typically less than 5%, more typically less than 4%, 2% or1%) in an encoded sequence are “conservatively modified variations”where the alterations result in the deletion of an amino acid, additionof an amino acid, or substitution of an amino acid with a chemicallysimilar amino acid. Thus, “conservative variations” of a listedpolypeptide sequence of the present invention include substitutions of asmall percentage, typically less than 5%, more typically less than 2% or1%, of the amino acids of the polypeptide sequence, with an amino acidof the same conservative substitution group. Finally, the addition ofsequences which do not alter the encoded activity of a nucleic acidmolecule, such as the addition of a non-functional sequence, is aconservative variation of the basic nucleic acid.

Conservative substitution tables providing functionally similar aminoacids are well known in the art, where one amino acid residue issubstituted for another amino acid residue having similar chemicalproperties (e.g., aromatic side chains or positively charged sidechains), and therefore does not substantially change the functionalproperties of the polypeptide molecule. The following sets forth examplegroups that contain natural amino acids of like chemical properties,where substitutions within a group is a “conservative substitution”.

TABLE 1 Conservative Amino Acid Substitutions Nonpolar and/or NegativelyAliphatic Polar, Positively Charged Side Uncharged Aromatic Charged SideSide Chains Side Chains Side Chains Chains Chains Glycine SerinePhenylalanine Lysine Aspartate Alanine Threonine Tyrosine ArginineGlutamate Valine Cysteine Tryptophan Histidine Leucine MethionineIsoleucine Asparagine Proline Glutamine

Nucleic Acid Hybridization

Comparative hybridization can be used to identify nucleic acids of theinvention, including conservative variations of nucleic acids of theinvention, and this comparative hybridization method is a preferredmethod of distinguishing nucleic acids of the invention. In addition,target nucleic acids which hybridize to a nucleic acid represented bySEQ ID NOS: 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39,41, 43, 45, 47, 51, 56 and 58, under high, ultra-high and ultra-ultrahigh stringency conditions are a feature of the invention. Examples ofsuch nucleic acids include those with one or a few silent orconservative nucleic acid substitutions as compared to a given nucleicacid sequence.

A test nucleic acid is said to specifically hybridize to a probe nucleicacid when it hybridizes at least 50% as well to the probe as to theperfectly matched complementary target, i.e., with a signal to noiseratio at least half as high as hybridization of the probe to the targetunder conditions in which the perfectly matched probe binds to theperfectly matched complementary target with a signal to noise ratio thatis at least about 5×-10× as high as that observed for hybridization toany of the unmatched target nucleic acids.

Nucleic acids “hybridize” when they associate, typically in solution.Nucleic acids hybridize due to a variety of well characterizedphysico-chemical forces, such as hydrogen bonding, solvent exclusion,base stacking and the like. An extensive guide to the hybridization ofnucleic acids is found in Tijssen (1993) Laboratory Techniques inBiochemistry and Molecular Biology—Hybridization with Nucleic AcidProbes part I chapter 2, “Overview of principles of hybridization andthe strategy of nucleic acid probe assays,” (Elsevier, N.Y.), as well asin Current Protocols in Molecular Biology, Ausubel et al., eds., CurrentProtocols, a joint venture between Greene Publishing Associates, Inc.and John Wiley & Sons, Inc., (supplemented through 2004) (“Ausubel”);Hames and Higgins (1995) Gene Probes 1 IRL Press at Oxford UniversityPress, Oxford, England, (Hames and Higgins 1) and Hames and Higgins(1995) Gene Probes 2 IRL Press at Oxford University Press, Oxford,England (Hames and Higgins 2) provide details on the synthesis,labeling, detection and quantification of DNA and RNA, includingoligonucleotides.

An example of stringent hybridization conditions for hybridization ofcomplementary nucleic acids which have more than 100 complementaryresidues on a filter in a Southern or northern blot is 50% formalin with1 mg of heparin at 42° C., with the hybridization being carried outovernight. An example of stringent wash conditions is a 0.2×SSC wash at65° C. for 15 minutes (see, Sambrook, supra for a description of SSCbuffer). Often the high stringency wash is preceded by a low stringencywash to remove background probe signal. An example low stringency washis 2×SSC at 40° C. for 15 minutes. In general, a signal to noise ratioof 5×(or higher) than that observed for an unrelated probe in theparticular hybridization assay indicates detection of a specifichybridization.

“Stringent hybridization wash conditions” in the context of nucleic acidhybridization experiments such as Southern and northern hybridizationsare sequence dependent, and are different under different environmentalparameters. An extensive guide to the hybridization of nucleic acids isfound in Tijssen (1993), supra. and in Hames and Higgins, 1 and 2.Stringent hybridization and wash conditions can easily be determinedempirically for any test nucleic acid. For example, in determiningstringent hybridization and wash conditions, the hybridization and washconditions are gradually increased (e.g., by increasing temperature,decreasing salt concentration, increasing detergent concentration and/orincreasing the concentration of organic solvents such as formalin in thehybridization or wash), until a selected set of criteria are met. Forexample, in highly stringent hybridization and wash conditions, thehybridization and wash conditions are gradually increased until a probebinds to a perfectly matched complementary target with a signal to noiseratio that is at least 5× as high as that observed for hybridization ofthe probe to an unmatched target.

“Very stringent” conditions are selected to be equal to the thermalmelting point (T_(m)) for a particular probe. The T_(m) is thetemperature (under defined ionic strength and pH) at which 50% of thetest sequence hybridizes to a perfectly matched probe. For the purposesof the present invention, generally, “highly stringent” hybridizationand wash conditions are selected to be about 5° C. lower than the T_(m)for the specific sequence at a defined ionic strength and pH.

“Ultra high-stringency” hybridization and wash conditions are those inwhich the stringency of hybridization and wash conditions are increaseduntil the signal to noise ratio for binding of the probe to theperfectly matched complementary target nucleic acid is at least 10× ashigh as that observed for hybridization to any of the unmatched targetnucleic acids. A target nucleic acid which hybridizes to a probe undersuch conditions, with a signal to noise ratio of at least ½ that of theperfectly matched complementary target nucleic acid is said to bind tothe probe under ultra-high stringency conditions.

Similarly, even higher levels of stringency can be determined bygradually increasing the hybridization and/or wash conditions of therelevant hybridization assay. For example, those in which the stringencyof hybridization and wash conditions are increased until the signal tonoise ratio for binding of the probe to the perfectly matchedcomplementary target nucleic acid is at least 10×, 20×, 50×, 100×, or500× or more as high as that observed for hybridization to any of theunmatched target nucleic acids. A target nucleic acid which hybridizesto a probe under such conditions, with a signal to noise ratio of atleast ½ that of the perfectly matched complementary target nucleic acidis said to bind to the probe under ultra-ultra-high stringencyconditions.

Nucleic acids which do not hybridize to each other under stringentconditions are still substantially identical if the polypeptides whichthey encode are substantially identical. This occurs, e.g., when a copyof a nucleic acid is created using the maximum codon degeneracypermitted by the genetic code.

Unique Subsequences

In some aspects, the invention provides a nucleic acid that comprises aunique subsequence in a nucleic acid selected from the sequences ofO-tRNAs and O-RSs disclosed herein. The unique subsequence is unique ascompared to a nucleic acid corresponding to any known O-tRNA or O-RSnucleic acid sequence. Alignment can be performed using, e.g., BLAST setto default parameters. Any unique subsequence is useful, e.g., as aprobe to identify the nucleic acids of the invention.

Similarly, the invention includes a polypeptide which comprises a uniquesubsequence in a polypeptide selected from the sequences of O-RSsdisclosed herein. Here, the unique subsequence is unique as compared toa polypeptide corresponding to any of known polypeptide sequence.

The invention also provides for target nucleic acids which hybridizesunder stringent conditions to a unique coding oligonucleotide whichencodes a unique subsequence in a polypeptide selected from thesequences of O-RSs wherein the unique subsequence is unique as comparedto a polypeptide corresponding to any of the control polypeptides (e.g.,parental sequences from which synthetases of the invention were derived,e.g., by mutation). Unique sequences are determined as noted above.

Sequence Comparison, Identity, and Homology

The terms “identical” or “percent identity,” in the context of two ormore nucleic acid or polypeptide sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same, whencompared and aligned for maximum correspondence, as measured using oneof the sequence comparison algorithms described below (or otheralgorithms available to persons of skill) or by visual inspection.

The phrase “substantially identical,” in the context of two nucleicacids or polypeptides (e.g., DNAs encoding an O-tRNA or O-RS, or theamino acid sequence of an O-RS) refers to two or more sequences orsubsequences that have at least about 60%, about 80%, about 90-95%,about 98%, about 99% or more nucleotide or amino acid residue identity,when compared and aligned for maximum correspondence, as measured usinga sequence comparison algorithm or by visual inspection. Such“substantially identical” sequences are typically considered to be“homologous,” without reference to actual ancestry. Preferably, the“substantial identity” exists over a region of the sequences that is atleast about 50 residues in length, more preferably over a region of atleast about 100 residues, and most preferably, the sequences aresubstantially identical over at least about 150 residues, or over thefull length of the two sequences to be compared.

Proteins and/or protein sequences are “homologous” when they arederived, naturally or artificially, from a common ancestral protein orprotein sequence. Similarly, nucleic acids and/or nucleic acid sequencesare homologous when they are derived, naturally or artificially, from acommon ancestral nucleic acid or nucleic acid sequence. For example, anynaturally occurring nucleic acid can be modified by any availablemutagenesis method to include one or more selector codon. Whenexpressed, this mutagenized nucleic acid encodes a polypeptidecomprising one or more unnatural amino acid. The mutation process can,of course, additionally alter one or more standard codon, therebychanging one or more standard amino acid in the resulting mutant proteinas well. Homology is generally inferred from sequence similarity betweentwo or more nucleic acids or proteins (or sequences thereof). Theprecise percentage of similarity between sequences that is useful inestablishing homology varies with the nucleic acid and protein at issue,but as little as 25% sequence similarity is routinely used to establishhomology. Higher levels of sequence similarity, e.g., 30%, 40%, 50%,60%, 70%, 80%, 90%, 95%, or 99% or more, can also be used to establishhomology. Methods for determining sequence similarity percentages (e.g.,BLASTP and BLASTN using default parameters) are described herein and aregenerally available.

For sequence comparison and homology determination, typically onesequence acts as a reference sequence to which test sequences arecompared. When using a sequence comparison algorithm, test and referencesequences are input into a computer, subsequence coordinates aredesignated, if necessary, and sequence algorithm program parameters aredesignated. The sequence comparison algorithm then calculates thepercent sequence identity for the test sequence(s) relative to thereference sequence, based on the designated program parameters.

Optimal alignment of sequences for comparison can be conducted, e.g., bythe local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482(1981), by the homology alignment algorithm of Needleman & Wunsch, J.Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson& Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis.), or by visual inspection (see generallyCurrent Protocols in Molecular Biology, Ausubel et al., eds., CurrentProtocols, a joint venture between Greene Publishing Associates, Inc.and John Wiley & Sons, Inc., supplemented through 2004).

One example of an algorithm that is suitable for determining percentsequence identity and sequence similarity is the BLAST algorithm, whichis described in Altschul et al., J. Mol. Biol. 215:403-410 (1990).Software for performing BLAST analyses is publicly available through theNational Center for Biotechnology Information (www.ncbi.nlm.nih.gov/).This algorithm involves first identifying high scoring sequence pairs(HSPs) by identifying short words of length W in the query sequence,which either match or satisfy some positive-valued threshold score Twhen aligned with a word of the same length in a database sequence. T isreferred to as the neighborhood word score threshold (Altschul et al.,supra). These initial neighborhood word hits act as seeds for initiatingsearches to find longer HSPs containing them. The word hits are thenextended in both directions along each sequence for as far as thecumulative alignment score can be increased. Cumulative scores arecalculated using, for nucleotide sequences, the parameters M (rewardscore for a pair of matching residues; always>0) and N (penalty scorefor mismatching residues; always<0). For amino acid sequences, a scoringmatrix is used to calculate the cumulative score. Extension of the wordhits in each direction are halted when: the cumulative alignment scorefalls off by the quantity X from its maximum achieved value; thecumulative score goes to zero or below, due to the accumulation of oneor more negative-scoring residue alignments; or the end of eithersequence is reached. The BLAST algorithm parameters W, T, and Xdetermine the sensitivity and speed of the alignment. The BLASTN program(for nucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) of 10, a cutoff of 100, M=5, N=−4, and a comparison ofboth strands. For amino acid sequences, the BLASTP program uses asdefaults a wordlength (W) of 3, an expectation (E) of 10, and theBLOSUM62 scoring matrix (see Henikoff & Henikoff (1989) Proc. Natl.Acad. Sci. USA 89:10915).

In addition to calculating percent sequence identity, the BLASTalgorithm also performs a statistical analysis of the similarity betweentwo sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA90:5873-5787 (1993)). One measure of similarity provided by the BLASTalgorithm is the smallest sum probability (P(N)), which provides anindication of the probability by which a match between two nucleotide oramino acid sequences would occur by chance. For example, a nucleic acidis considered similar to a reference sequence if the smallest sumprobability in a comparison of the test nucleic acid to the referencenucleic acid is less than about 0.1, more preferably less than about0.01, and most preferably less than about 0.001.

Mutagenesis and Other Molecular Biology Techniques

Polynucleotide and polypeptides of the invention and used in theinvention can be manipulated using molecular biological techniques.General texts which describe molecular biological techniques includeBerger and Kimmel, Guide to Molecular Cloning Techniques, Methods inEnzymology volume 152 Academic Press, Inc., San Diego, Calif. (Berger);Sambrook et al., Molecular Cloning—A Laboratory Manual (3rd Ed.), Vol.1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 2001(“Sambrook”) and Current Protocols in Molecular Biology, F. M. Ausubelet al., eds., Current Protocols, a joint venture between GreenePublishing Associates, Inc. and John Wiley & Sons, Inc., (supplementedthrough 2004) (“Ausubel”). These texts describe mutagenesis, the use ofvectors, promoters and many other relevant topics related to, e.g., thegeneration of genes that include selector codons for production ofproteins that include unnatural amino acids, orthogonal tRNAs,orthogonal synthetases, and pairs thereof.

Various types of mutagenesis are used in the invention, e.g., to mutatetRNA molecules, to produce libraries of tRNAs, to produce libraries ofsynthetases, to insert selector codons that encode an unnatural aminoacids in a protein or polypeptide of interest. They include but are notlimited to site-directed, random point mutagenesis, homologousrecombination, DNA shuffling or other recursive mutagenesis methods,chimeric construction, mutagenesis using uracil containing templates,oligonucleotide-directed mutagenesis, phosphorothioate-modified DNAmutagenesis, mutagenesis using gapped duplex DNA or the like, or anycombination thereof. Additional suitable methods include point mismatchrepair, mutagenesis using repair-deficient host strains,restriction-selection and restriction-purification, deletionmutagenesis, mutagenesis by total gene synthesis, double-strand breakrepair, and the like. Mutagenesis, e.g., involving chimeric constructs,is also included in the present invention. In one embodiment,mutagenesis can be guided by known information of the naturallyoccurring molecule or altered or mutated naturally occurring molecule,e.g., sequence, sequence comparisons, physical properties, crystalstructure or the like.

Host cells are genetically engineered (e.g., transformed, transduced ortransfected) with the polynucleotides of the invention or constructswhich include a polynucleotide of the invention, e.g., a vector of theinvention, which can be, for example, a cloning vector or an expressionvector. For example, the coding regions for the orthogonal tRNA, theorthogonal tRNA synthetase, and the protein to be derivatized areoperably linked to gene expression control elements that are functionalin the desired host cell. Typical vectors contain transcription andtranslation terminators, transcription and translation initiationsequences, and promoters useful for regulation of the expression of theparticular target nucleic acid. The vectors optionally comprise genericexpression cassettes containing at least one independent terminatorsequence, sequences permitting replication of the cassette ineukaryotes, or prokaryotes, or both (e.g., shuttle vectors) andselection markers for both prokaryotic and eukaryotic systems. Vectorsare suitable for replication and/or integration in prokaryotes,eukaryotes, or preferably both. See Giliman & Smith, Gene 8:81 (1979);Roberts, et al., Nature, 328:731 (1987); Schneider, B., et al., ProteinExpr. Purif. 6435:10 (1995); Ausubel, Sambrook, Berger (all supra). Thevector can be, for example, in the form of a plasmid, a bacterium, avirus, a naked polynucleotide, or a conjugated polynucleotide. Thevectors are introduced into cells and/or microorganisms by standardmethods including electroporation (From et al., Proc. Natl. Acad. Sci.USA 82, 5824 (1985), infection by viral vectors, high velocity ballisticpenetration by small particles with the nucleic acid either within thematrix of small beads or particles, or on the surface (Klein et al.,Nature 327, 70-73 (1987)), and/or the like.

A highly efficient and versatile single plasmid system was developed forsite-specific incorporation of unnatural amino acids into proteins inresponse to the amber stop codon (UAG) in E. coli. In the new system,the pair of M. jannaschii suppressor tRNAtyr(CUA) and tyrosyl-tRNAsynthetase are encoded in a single plasmid, which is compatible withmost E. coli expression vectors. Monocistronic tRNA operon under controlof proK promoter and terminator was constructed for optimal secondarystructure and tRNA processing. Introduction of a mutated form of glnSpromoter for the synthetase resulted in a significant increase in bothsuppression efficiency and fidelity. Increases in suppression efficiencywere also obtained by multiple copies of tRNA gene as well as by aspecific mutation (D286R) on the synthetase (Kobayashi et al.,“Structural basis for orthogonal tRNA specificities of tyrosyl-tRNAsynthetases for genetic code expansion,” Nat. Struct. Biol.,10(6):425-432 [2003]). The generality of the optimized system was alsodemonstrated by highly efficient and accurate incorporation of severaldifferent unnatural amino acids, whose unique utilities in studyingprotein function and structure were previously proven.

A catalogue of Bacteria and Bacteriophages useful for cloning isprovided, e.g., by the ATCC, e.g., The ATCC Catalogue of Bacteria andBacteriophage (1996) Gherna et al. (eds) published by the ATCC.Additional basic procedures for sequencing, cloning and other aspects ofmolecular biology and underlying theoretical considerations are alsofound in Sambrook (supra), Ausubel (supra), and in Watson et al. (1992)Recombinant DNA Second Edition Scientific American Books, NY. Inaddition, essentially any nucleic acid (and virtually any labelednucleic acid, whether standard or non-standard) can be custom orstandard ordered from any of a variety of commercial sources, such asthe Midland Certified Reagent Company (Midland, Tex. mcrc.com), TheGreat American Gene Company (Ramona, Calif. available on the World WideWeb at genco.com), ExpressGen Inc. (Chicago, Ill. available on the WorldWide Web at expressgen.com), Operon Technologies Inc. (Alameda, Calif.)and many others.

The engineered host cells can be cultured in conventional nutrient mediamodified as appropriate for such activities as, for example, screeningsteps, activating promoters or selecting transformants. These cells canoptionally be cultured into transgenic organisms. Other usefulreferences, e.g. for cell isolation and culture (e.g., for subsequentnucleic acid isolation) include Freshney (1994) Culture of Animal Cells,a Manual of Basic Technique, third edition, Wiley-Liss, New York and thereferences cited therein; Payne et al. (1992) Plant Cell and TissueCulture in Liquid Systems John Wiley & Sons, Inc. New York, N.Y.;Gamborg and Phillips (eds) (1995) Plant Cell, Tissue and Organ Culture;Fundamental Methods Springer Lab Manual, Springer-Verlag (BerlinHeidelberg New York) and Atlas and Parks (eds) The Handbook ofMicrobiological Media (1993) CRC Press, Boca Raton, Fla.

Proteins and Polypeptides of Interest

Methods of producing a protein in a cell with an unnatural amino acid ata specified position are also a feature of the invention. For example, amethod includes growing, in an appropriate medium, the cell, where thecell comprises a nucleic acid that comprises at least one selector codonand encodes a protein; and, providing the unnatural amino acid; wherethe cell further comprises: an orthogonal-tRNA (O-tRNA) that functionsin the cell and recognizes the selector codon; and, an orthogonalaminoacyl-tRNA synthetase (O-RS) that preferentially aminoacylates theO-tRNA with the unnatural amino acid. A protein produced by this methodis also a feature of the invention.

In certain embodiments, the O-RS comprises a bias for the aminoacylationof the cognate O-tRNA over any endogenous tRNA in an expression system.The relative ratio between O-tRNA and endogenous tRNA that is charged bythe O-RS, when the O-tRNA and O-RS are present at equal molarconcentrations, is greater than 1:1, preferably at least about 2:1, morepreferably 5:1, still more preferably 10:1, yet more preferably 20:1,still more preferably 50:1, yet more preferably 75:1, still morepreferably 95:1, 98:1, 99:1, 100:1, 500:1, 1, 000:1, 5, 000:1 or higher.

The invention also provides compositions that include proteins, wherethe proteins comprise an unnatural amino acid. In certain embodiments,the protein comprises an amino acid sequence that is at least 75%identical to that of a therapeutic protein, a diagnostic protein, anindustrial enzyme, or portion thereof.

The compositions of the invention and compositions made by the methodsof the invention optionally are in a cell. The O-tRNA/O-RS pairs orindividual components of the invention can then be used in a hostsystem's translation machinery, which results in an unnatural amino acidbeing incorporated into a protein. International Publication Numbers WO2004/094593, filed Apr. 16, 2004, entitled “EXPANDING THE EUKARYOTICGENETIC CODE,” and WO 2002/085923, entitled “IN VIVO INCORPORATION OFUNNATURAL AMINO ACIDS,” describe this process, and are incorporatedherein by reference. For example, when an O-tRNA/O-RS pair is introducedinto a host, e.g., an Escherichia coli cell or a yeast cell, the pairleads to the in vivo incorporation of an unnatural amino acid such asp-ethylthiocarbonyl-L-phenylalanine, p-(3-oxobutanoyl)-L-phenylalanine,1,5-dansyl-alanine, 7-amino-coumarin alanine, 7-hydroxy-coumarinalanine, o-nitrobenzyl-serine, O-(2-nitrobenzyl)-L-tyrosine,p-carboxymethyl-L-phenylalanine, p-cyano-L-phenylalanine,m-cyano-L-phenylalanine, biphenylalanine, 3-amino-L-tyrosine,bipyridylalanine, p-(2-amino-1-hydroxyethyl)-L-phenylalanine;p-isopropylthiocarbonyl-L-phenylalanine; 3-nitro-L-tyrosine orp-nitro-L-phenylalanine into a protein in response to a selector codon.The unnatural amino acid that is added to the system can a syntheticamino acid, such as a derivative of a phenylalanine or tyrosine, whichcan be exogenously added to the growth medium. Optionally, thecompositions of the present invention can be in an in vitro translationsystem, or in an in vivo system(s).

A cell of the invention provides the ability to synthesize proteins thatcomprise unnatural amino acids in large useful quantities. In someaspects, the composition optionally includes, e.g., at least 10micrograms, at least 50 micrograms, at least 75 micrograms, at least 100micrograms, at least 200 micrograms, at least 250 micrograms, at least500 micrograms, at least 1 milligram, at least 10 milligrams or more ofthe protein that comprises an unnatural amino acid, or an amount thatcan be achieved with in vivo protein production methods (details onrecombinant protein production and purification are provided herein). Inanother aspect, the protein is optionally present in the composition ata concentration of, e.g., at least 10 micrograms of protein per liter,at least 50 micrograms of protein per liter, at least 75 micrograms ofprotein per liter, at least 100 micrograms of protein per liter, atleast 200 micrograms of protein per liter, at least 250 micrograms ofprotein per liter, at least 500 micrograms of protein per liter, atleast 1 milligram of protein per liter, or at least 10 milligrams ofprotein per liter or more, in, e.g., a cell lysate, a buffer, apharmaceutical buffer, or other liquid suspension (e.g., in a volume of,e.g., anywhere from about 1 mL to about 100 L). The production of largequantities (e.g., greater that that typically possible with othermethods, e.g., in vitro translation) of a protein in a cell including atleast one unnatural amino acid is a feature of the invention.

The incorporation of an unnatural amino acid can be done to, e.g.,tailor changes in protein structure and/or function, e.g., to changesize, acidity, nucleophilicity, hydrogen bonding, hydrophobicity,accessibility of protease target sites, target to a moiety (e.g., for aprotein array), incorporation of labels or reactive groups, etc.Proteins that include an unnatural amino acid can have enhanced or evenentirely new catalytic or physical properties. For example, thefollowing properties are optionally modified by inclusion of anunnatural amino acid into a protein: toxicity, biodistribution,structural properties, spectroscopic properties, chemical and/orphotochemical properties, catalytic ability, half-life (e.g., serumhalf-life), ability to react with other molecules, e.g., covalently ornoncovalently, and the like. The compositions including proteins thatinclude at least one unnatural amino acid are useful for, e.g., noveltherapeutics, diagnostics, catalytic enzymes, industrial enzymes,binding proteins (e.g., antibodies), and e.g., the study of proteinstructure and function. See, e.g., Dougherty, (2000) Unnatural AminoAcids as Probes of Protein Structure and Function, Current Opinion inChemical Biology, 4:645-652.

In some aspects of the invention, a composition includes at least oneprotein with at least one, e.g., at least two, at least three, at leastfour, at least five, at least six, at least seven, at least eight, atleast nine, or at least ten or more unnatural amino acids. The unnaturalamino acids can be the same or different, e.g., there can be 1, 2, 3, 4,5, 6, 7, 8, 9, or 10 or more different sites in the protein thatcomprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more different unnaturalamino acids. In another aspect, a composition includes a protein with atleast one, but fewer than all, of a particular amino acid present in theprotein is an unnatural amino acid. For a given protein with more thanone unnatural amino acids, the unnatural amino acids can be identical ordifferent (e.g., the protein can include two or more different types ofunnatural amino acids, or can include two of the same unnatural aminoacid). For a given protein with more than two unnatural amino acids, theunnatural amino acids can be the same, different or a combination of amultiple unnatural amino acid of the same kind with at least onedifferent unnatural amino acid.

Essentially any protein (or portion thereof) that includes an unnaturalamino acid (and any corresponding coding nucleic acid, e.g., whichincludes one or more selector codons) can be produced using thecompositions and methods herein. No attempt is made to identify thehundreds of thousands of known proteins, any of which can be modified toinclude one or more unnatural amino acid, e.g., by tailoring anyavailable mutation methods to include one or more appropriate selectorcodon in a relevant translation system. Common sequence repositories forknown proteins include GenBank EMBL, DDBJ and the NCBI. Otherrepositories can easily be identified by searching the interne.

Typically, the proteins are, e.g., at least 60%, at least 70%, at least75%, at least 80%, at least 90%, at least 95%, or at least 99% or moreidentical to any available protein (e.g., a therapeutic protein, adiagnostic protein, an industrial enzyme, or portion thereof, and thelike), and they comprise one or more unnatural amino acid. Examples oftherapeutic, diagnostic, and other proteins that can be modified tocomprise one or more unnatural amino acid can be found, but not limitedto, those in International Publications WO 2004/094593, filed Apr. 16,2004, entitled “Expanding the Eukaryotic Genetic Code;” and, WO2002/085923, entitled “IN VIVO INCORPORATION OF UNNATURAL AMINO ACIDS.”Examples of therapeutic, diagnostic, and other proteins that can bemodified to comprise one or more unnatural amino acids include, but arenot limited to, e.g., Alpha-1 antitrypsin, Angiostatin, Antihemolyticfactor, antibodies (further details on antibodies are found below),Apolipoprotein, Apoprotein, Atrial natriuretic factor, Atrialnatriuretic polypeptide, Atrial peptides, C—X—C chemokines (e.g.,T39765, NAP-2, ENA-78, Gro-a, Gro-b, Gro-c, IP-10, GCP-2, NAP-4, SDF-1,PF4, MIG), Calcitonin, CC chemokines (e.g., Monocyte chemoattractantprotein-1, Monocyte chemoattractant protein-2, Monocyte chemoattractantprotein-3, Monocyte inflammatory protein-1 alpha, Monocyte inflammatoryprotein-1 beta, RANTES, 1309, R83915, R91733, HCC1, T58847, D31065,T64262), CD40 ligand, C-kit Ligand, Collagen, Colony stimulating factor(CSF), Complement factor 5a, Complement inhibitor, Complement receptor1, cytokines, (e.g., epithelial Neutrophil Activating Peptide-78,GROα/MGSA, GROβ, GROγ, MIP-1α, MIP-1δ, MCP-1), Epidermal Growth Factor(EGF), Erythropoietin (“EPO”), Exfoliating toxins A and B, Factor IX,Factor VII, Factor VIII, Factor X, Fibroblast Growth Factor (FGF),Fibrinogen, Fibronectin, G-CSF, GM-CSF, Glucocerebrosidase,Gonadotropin, growth factors, Hedgehog proteins (e.g., Sonic, Indian,Desert), Hemoglobin, Hepatocyte Growth Factor (HGF), Hirudin, Humanserum albumin, Insulin, Insulin-like Growth Factor (IGF), interferons(e.g., IFN-α, IFN-β, IFN-γ), interleukins (e.g., IL-1, IL-2, IL-3, IL-4,IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, etc.), KeratinocyteGrowth Factor (KGF), Lactoferrin, leukemia inhibitory factor,Luciferase, Neurturin, Neutrophil inhibitory factor (NIF), oncostatin M,Osteogenic protein, Parathyroid hormone, PD-ECSF, PDGF, peptide hormones(e.g., Human Growth Hormone), Pleiotropin, Protein A, Protein G,Pyrogenic exotoxins A, B, and C, Relaxin, Renin, SCF, Soluble complementreceptor I, Soluble I-CAM 1, Soluble interleukin receptors (IL-1,2,3,4,5, 6, 7, 9, 10, 11, 12, 13, 14, 15), Soluble TNF receptor, Somatomedin,Somatostatin, Somatotropin, Streptokinase, Superantigens, i.e.,Staphylococcal enterotoxins (SEA, SEB, SEC1, SEC2, SEC3, SED, SEE),Superoxide dismutase (SOD), Toxic shock syndrome toxin (TSST-1),Thymosin alpha 1, Tissue plasminogen activator, Tumor necrosis factorbeta (TNF beta), Tumor necrosis factor receptor (TNFR), Tumor necrosisfactor-alpha (TNF alpha), Vascular Endothelial Growth Factor (VEGEF),Urokinase and many others.

One class of proteins that can be made using the compositions andmethods for in vivo incorporation of unnatural amino acids describedherein includes transcriptional modulators or a portion thereof. Exampletranscriptional modulators include genes and transcriptional modulatorproteins that modulate cell growth, differentiation, regulation, or thelike. Transcriptional modulators are found in prokaryotes, viruses, andeukaryotes, including fungi, plants, yeasts, insects, and animals,including mammals, providing a wide range of therapeutic targets. Itwill be appreciated that expression and transcriptional activatorsregulate transcription by many mechanisms, e.g., by binding toreceptors, stimulating a signal transduction cascade, regulatingexpression of transcription factors, binding to promoters and enhancers,binding to proteins that bind to promoters and enhancers, unwinding DNA,splicing pre-mRNA, polyadenylating RNA, and degrading RNA.

One class of proteins of the invention (e.g., proteins with one or moreunnatural amino acids) include biologically active proteins such ascytokines, inflammatory molecules, growth factors, their receptors, andoncogene products, e.g., interleukins (e.g., IL-1, IL-2, IL-8, etc.),interferons, FGF, IGF-I, IGF-II, FGF, PDGF, TNF, TGF-α, TGF-β, EGF, KGF,SCF/c-Kit, CD40L/CD40, VLA-4/VCAM-1, ICAM-1/LFA-1, and hyalurin/CD44;signal transduction molecules and corresponding oncogene products, e.g.,Mos, Ras, Raf, and Met; and transcriptional activators and suppressors,e.g., p53, Tat, Fos, Myc, Jun, Myb, Rel, and steroid hormone receptorssuch as those for estrogen, progesterone, testosterone, aldosterone, theLDL receptor ligand and corticosterone.

Enzymes (e.g., industrial enzymes) or portions thereof with at least oneunnatural amino acid are also provided by the invention. Examples ofenzymes include, but are not limited to, e.g., amidases, amino acidracemases, acylases, dehalogenases, dioxygenases, diarylpropaneperoxidases, epimerases, epoxide hydrolases, esterases, isomerases,kinases, glucose isomerases, glycosidases, glycosyl transferases,haloperoxidases, monooxygenases (e.g., p450s), lipases, ligninperoxidases, nitrile hydratases, nitrilases, proteases, phosphatases,subtilisins, transaminase, and nucleases.

Many of these proteins are commercially available (See, e.g., the SigmaBioSciences 2002 catalogue and price list), and the correspondingprotein sequences and genes and, typically, many variants thereof, arewell-known (see, e.g., Genbank). Any of them can be modified by theinsertion of one or more unnatural amino acid according to theinvention, e.g., to alter the protein with respect to one or moretherapeutic, diagnostic or enzymatic properties of interest. Examples oftherapeutically relevant properties include serum half-life, shelfhalf-life, stability, immunogenicity, therapeutic activity,detectability (e.g., by the inclusion of reporter groups (e.g., labelsor label binding sites) in the unnatural amino acids), reduction of LD₅₀or other side effects, ability to enter the body through the gastrictract (e.g., oral availability), or the like. Examples of diagnosticproperties include shelf half-life, stability, diagnostic activity,detectability, or the like. Examples of relevant enzymatic propertiesinclude shelf half-life, stability, enzymatic activity, productioncapability, or the like.

A variety of other proteins can also be modified to include one or moreunnatural amino acid using compositions and methods of the invention.For example, the invention can include substituting one or more naturalamino acids in one or more vaccine proteins with an unnatural aminoacid, e.g., in proteins from infectious fungi, e.g., Aspergillus,Candida species; bacteria, particularly E. coli, which serves a modelfor pathogenic bacteria, as well as medically important bacteria such asStaphylococci (e.g., aureus), or Streptococci (e.g., pneumoniae);protozoa such as sporozoa (e.g., Plasmodia), rhizopods (e.g., Entamoeba)and flagellates (Trypanosoma, Leishmania, Trichomonas, Giardia, etc.);viruses such as (+) RNA viruses (examples include Poxviruses e.g.,vaccinia; Picornaviruses, e.g. polio; Togaviruses, e.g., rubella;Flaviviruses, e.g., HCV; and Coronaviruses), (−) RNA viruses (e.g.,Rhabdoviruses, e.g., VSV; Paramyxovimses, e.g., RSV; Orthomyxovimses,e.g., influenza; Bunyaviruses; and Arenaviruses), dsDNA viruses(Reoviruses, for example), RNA to DNA viruses, i.e., Retroviruses, e.g.,HIV and HTLV, and certain DNA to RNA viruses such as Hepatitis B.

Agriculturally related proteins such as insect resistance proteins(e.g., the Cry proteins), starch and lipid production enzymes, plant andinsect toxins, toxin-resistance proteins, Mycotoxin detoxificationproteins, plant growth enzymes (e.g., Ribulose 1,5-BisphosphateCarboxylase/Oxygenase, “RUBISCO”), lipoxygenase (LOX), andPhosphoenolpyruvate (PEP) carboxylase are also suitable targets forunnatural amino acid modification.

In certain embodiments, the protein or polypeptide of interest (orportion thereof) in the methods and/or compositions of the invention isencoded by a nucleic acid. Typically, the nucleic acid comprises atleast one selector codon, at least two selector codons, at least threeselector codons, at least four selector codons, at least five selectorcodons, at least six selector codons, at least seven selector codons, atleast eight selector codons, at least nine selector codons, ten or moreselector codons.

Genes coding for proteins or polypeptides of interest can be mutagenizedusing methods well-known to one of skill in the art and described hereinunder “Mutagenesis and Other Molecular Biology Techniques” to include,e.g., one or more selector codon for the incorporation of an unnaturalamino acid. For example, a nucleic acid for a protein of interest ismutagenized to include one or more selector codon, providing for theinsertion of the one or more unnatural amino acids. The inventionincludes any such variant, e.g., mutant, versions of any protein, e.g.,including at least one unnatural amino acid. Similarly, the inventionalso includes corresponding nucleic acids, i.e., any nucleic acid withone or more selector codon that encodes one or more unnatural aminoacid.

To make a protein that includes an unnatural amino acid, one can usehost cells and organisms that are adapted for the in vivo incorporationof the unnatural amino acid via orthogonal tRNA/RS pairs. Host cells aregenetically engineered (e.g., transformed, transduced or transfected)with one or more vectors that express the orthogonal tRNA, theorthogonal tRNA synthetase, and a vector that encodes the protein to bederivatized. Each of these components can be on the same vector, or eachcan be on a separate vector, or two components can be on one vector andthe third component on a second vector. The vector can be, for example,in the form of a plasmid, a bacterium, a virus, a naked polynucleotide,or a conjugated polynucleotide.

Defining Polypeptides by Immunoreactivity

Because the polypeptides of the invention provide a variety of newpolypeptide sequences (e.g., polypeptides comprising unnatural aminoacids in the case of proteins synthesized in the translation systemsherein, or, e.g., in the case of the novel synthetases, novel sequencesof standard amino acids), the polypeptides also provide new structuralfeatures which can be recognized, e.g., in immunological assays. Thegeneration of antisera, which specifically bind the polypeptides of theinvention, as well as the polypeptides which are bound by such antisera,are a feature of the invention. The term “antibody,” as used herein,includes, but is not limited to a polypeptide substantially encoded byan immunoglobulin gene or immunoglobulin genes, or fragments thereofwhich specifically bind and recognize an analyte (antigen). Examplesinclude polyclonal, monoclonal, chimeric, and single chain antibodies,and the like. Fragments of immunoglobulins, including Fab fragments andfragments produced by an expression library, including phage display,are also included in the term “antibody” as used herein. See, e.g.,Paul, Fundamental Immunology, 4th Ed., 1999, Raven Press, New York, forantibody structure and terminology.

In order to produce antisera for use in an immunoassay, one or more ofthe immunogenic polypeptides is produced and purified as describedherein. For example, recombinant protein can be produced in arecombinant cell. An inbred strain of mice (used in this assay becauseresults are more reproducible due to the virtual genetic identity of themice) is immunized with the immunogenic protein(s) in combination with astandard adjuvant, such as Freund's adjuvant, and a standard mouseimmunization protocol (see, e.g., Harlow and Lane (1988) Antibodies, ALaboratory Manual, Cold Spring Harbor Publications, New York, for astandard description of antibody generation, immunoassay formats andconditions that can be used to determine specific immunoreactivity.Additional details on proteins, antibodies, antisera, etc. can be foundin International Publication Numbers WO 2004/094593, entitled “EXPANDINGTHE EUKARYOTIC GENETIC CODE;” WO 2002/085923, entitled “IN VIVOINCORPORATION OF UNNATURAL AMINO ACIDS;” WO 2004/035605, entitled“GLYCOPROTEIN SYNTHESIS;” and WO 2004/058946, entitled “PROTEIN ARRAYS.”

Use of O-tRNA and O-RS and O-tRNA/O-RS Pairs

The compositions of the invention and compositions made by the methodsof the invention optionally are in a cell. The O-tRNA/O-RS pairs orindividual components of the invention can then be used in a hostsystem's translation machinery, which results in an unnatural amino acidbeing incorporated into a protein. International Publication Number WO2002/085923 by Schultz, et al., entitled “IN VIVO INCORPORATION OFUNNATURAL AMINO ACIDS,” describes this process and is incorporatedherein by reference. For example, when an O-tRNA/O-RS pair is introducedinto a host, e.g., Escherichia coli or yeast, the pair leads to the invivo incorporation of an unnatural amino acid, which can be exogenouslyadded to the growth medium, into a protein, e.g., a myoglobin testprotein or a therapeutic protein, in response to a selector codon, e.g.,an amber nonsense codon. Optionally, the compositions of the inventioncan be in an in vitro translation system, or in a cellular in vivosystem(s). Proteins with the unnatural amino acid can be used in any ofa wide range of applications. For example, the unnatural moietyincorporated into a protein can serve as a target for any of a widerange of modifications, for example, crosslinking with other proteins,with small molecules such as labels or dyes and/or biomolecules. Withthese modifications, incorporation of the unnatural amino acid canresult in improved therapeutic proteins and can be used to alter orimprove the catalytic function of enzymes. In some aspects, theincorporation and subsequent modification of an unnatural amino acid ina protein can facilitate studies on protein structure, interactions withother proteins, and the like.

Photoregulation and Photocaging

Photoregulated amino acids (e.g., photochromic, photocleavable,photoisomerizable, etc.) can be used to spatially and temporally controla variety of biological process, e.g., by directly regulating theactivity of enzymes, receptors, ion channels or the like, or bymodulating the intracellular concentrations of various signalingmolecules. See, e.g., Shigeri et al., Pharmacol. Therapeut., 2001,91:85; Curley, et al., Pharmacol. Therapeut., 1999, 82:347; Curley, etal., Curr. Op. Chem. Bio., 1999, 3:84; “Caged Compounds” Methods inEnzymology, Marriott, G., Ed, Academic Press, NY, 1998, V. 291; Adams,et al., Annu. Rev. Physiol., 1993, 55:755+; and Bochet, et al., J. Chem.Soc., Perkin 1, 2002, 125. In various embodiments herein, thecompositions and methods comprise photoregulated amino acids. Forexample, the invention provides orthogonal translation systems for theincorporation of the photoregulated unnatural amino acidso-nitrobenzyl-serine and O-(2-nitrobenzyl)-L-tyrosine (see, FIG. 1, andExamples 8 and 9).

“Photoregulated amino acids” are typically, e.g., photosensitive aminoacids. Photoregulated amino acids in general are those that arecontrolled in some fashion by light (e.g., UV, IR, etc.). Thus, forexample, if a photoregulated amino acid is incorporated into apolypeptide having biological activity, illumination can alter the aminoacid, thereby changing the biological activity of the peptide. Somephotoregulated amino acids can comprise “photocaged amino acids,”“photosensitive amino acids,” “photolabile amino acids,”“photoisomerizable,” etc. “Caged species,” such as caged amino acids, orcaged peptides, are those trapped inside a larger entity (e.g.,molecule) and that are released upon specific illumination. See, e.g.,Adams, et al., Annu. Rev. Physiol., 1993, 55:755-784. “Caging” groups ofamino acids can inhibit or conceal (e.g., by disrupting bonds whichwould usually stabilize interactions with target molecules, by changingthe hydrophobicity or ionic character of a particular side chain, or bysteric hindrance, etc.) biological activity in a molecule, e.g., apeptide comprising such amino acid. “Photoisomerizable” amino acids canswitch isomer forms due to light exposure. The different isomers of suchamino acids can end up having different interactions with other sidechains in a protein upon incorporation. Photoregulated amino acids canthus control the biological activity (either through activation, partialactivation, inactivation, partial inactivation, modified activation,etc.) of the peptides in which they are present. See Adams above andother references in this section for further definitions and examples ofphotoregulated amino acids and molecules.

A number of photoregulated amino acids are known to those in the art andmany are available commercially. Methods of attaching and/or associatingphotoregulating moieties to amino acids are also known. Suchphotoregulated amino acids in general are amenable to variousembodiments herein. It will be appreciated that while a number ofpossible photoregulating moieties, e.g., photocaging groups and thelike, as well as a number of photoregulated amino acids are listedherein, such recitation should not be taken as limiting. Thus, thecurrent invention is also amenable to photoregulating moieties andphotoregulated amino acids that are not specifically recited herein.

As stated, a number of methods are optionally applicable to create aphotoregulated amino acid. Thus, for example, a photoregulated aminoacid, e.g., a photocaged amino acid can be created by protecting itsα-amino group with compounds such as BOC (butyloxycarbonyl), andprotecting the α-carboxyl group with compounds such as a t-butyl ester.Such protection can be followed by reaction of the amino acid side chainwith a photolabile caging group such as 2-nitrobenzyl, in a reactiveform such as 2-nitrobenzylchloroformate, α-carboxyl 2-nitrobenzylbromide methyl ester, or 2-nitrobenzyl diazoethanc. After thephotolabile cage group is added, the protecting groups can be removedvia standard procedures. See, e.g., U.S. Pat. No. 5,998,580.

As another example, lysine residues can be caged using2-nitrobenzylchloroformate to derivatize the E-lysine amino group, thuseliminating the positive charge. Alternatively, lysine can be caged byintroducing a negative charge into a peptide (which has such lysine) byuse of an α-carboxy 2-nitrobenzyloxycarbonyl caging group. Additionally,phosphoserine and phosphothreonine can be caged by treatment of thephosphoamino acid or the phosphopeptide with1(2-nitrophenyl)diazoethane. See, e.g., Walker et al., Meth Enzymol.172:288-301, 1989. A number of other amino acids are also easilyamenable to standard caging chemistry, for example serine, threonine,histidine, glutamine, asparagine, aspartic acid and glutamic acid. See,e.g., Wilcox et al., J. Org. Chem. 55:1585-1589, 1990). Again, it willbe appreciated that recitation of particular photoregulated (amino acidsand/or those capable of being converted to photoregulated forms) shouldnot necessarily be taken as limiting.

Amino acid residues can also be made photoregulated (e.g.,photosensitive or photolabile) in other fashions. For example, certainamino acid residues can be created wherein irradiation causes cleavageof a peptide backbone that has the particular amino acid residue. Forexample a photolabile glycine, 2-nitrophenyl glycine, can function insuch a manner. See, e.g., Davis, et al., 1973, J. Med. Chem.,16:1043-1045. Irradiation of peptides containing 2-nitrophenylglycinewill cleave the peptide backbone between the alpha carbon and the alphaamino group of 2-nitrophenylglycine. Such cleavage strategy is generallyapplicable to amino acids other than glycine, if the 2-nitrobenzyl groupis inserted between the alpha carbon and the alpha amino group.

A large number of photoregulating groups, e.g., caging groups, and anumber of reactive compounds used to covalently attach such groups toother molecules such as amino acids, are well known in the art. Examplesof photoregulating (e.g., photolabile, caging) groups include, but arenot limited to: o-nitrobenzyl-serine, O-(2-nitrobenzyl)-L-tyrosine,nitroindolines; N-acyl-7-nitroindolines; phenacyls; hydroxyphenacyl;brominated 7-hydroxycoumarin-4-ylmethyls (e.g., Bhc); benzoin esters;dimethoxybenzoin; meta-phenols; 2-nitrobenzyl;1-(4,5-dimethoxy-2-nitrophenyl)ethyl (DMNPE);4,5-dimethoxy-2-nitrobenzyl (DMNB); alpha-carboxy-2-nitrobenzyl (CNB);1-(2-nitrophenyl)ethyl (NPE); 5-carboxymethoxy-2-nitrobenzyl (CMNB);(5-carboxymethoxy-2-nitrobenzyl)oxy) carbonyl;(4,5-dimethoxy-2-nitrobenzyl)oxy)carbonyl; desoxybenzoinyl; and thelike. See, e.g., U.S. Pat. No. 5,635,608 to Haugland and Gee (Jun. 3,1997) entitled “α-carboxy caged compounds” Neuro 19, 465 (1997); JPhysiol 508.3, 801 (1998); Proc Natl Acad Sci USA 1988 September,85(17):6571-5; J Biol Chem 1997 February 14, 272(7):4172-8; Neuron 20,619-624, 1998; Nature Genetics, vol. 28:2001:317-325; Nature, vol. 392,1998:936-941; Pan, P., and Bayley, H. “Caged cysteine and thiophosphorylpeptides” FEBS Letters 405:81-85 (1997); Pettit et al. (1997) “Chemicaltwo-photon uncaging: a novel approach to mapping glutamate receptors”Neuron 19:465-471; Furuta et al. (1999) “Brominated7-hydroxycoumarin-4-ylmethyls: novel photolabile protecting groups withbiologically useful cross-sections for two photon photolysis” Proc.Natl. Acad. Sci. 96(4):1193-1200; Zou et al. “Catalytic subunit ofprotein kinase A caged at the activating phosphothreonine” J. Amer.Chem. Soc. (2002) 124:8220-8229; Zou et al. “Caged ThiophosphotyrosinePeptides” Angew. Chem. Int. Ed. (2001) 40:3049-3051; Conrad I I et al.“p-Hydroxyphenacyl Phototriggers: The reactive Excited State ofPhosphate Photorelease” J. Am. Chem. Soc. (2000) 122:9346-9347; Conrad II et al. “New Phototriggers 10: Extending the π,π* Absorption to ReleasePeptides in Biological Media” Org. Lett. (2000) 2:1545-1547; Givens etal. “A New Phototriggers 9: p-Hydroxyphenacyl as a C-TerminusPhotoremovable Protecting Group for Oligopeptides” J. Am. Chem. Soc.(2000) 122:2687-2697; Bishop et al.“40-Aminomethyl-2,20-bipyridyl-4-carboxylic Acid (Abc) and RelatedDerivatives: Novel Bipyridine Amino Acids for the Solid-PhaseIncorporation of a Metal Coordination Site Within a Peptide Backbone”Tetrahedron (2000) 56:4629-4638; Ching et al. “Polymers As Surface-BasedTethers with Photolytic triggers Enabling Laser-InducedRelease/Desorption of Covalently Bound Molecules” Bioconjugate Chemistry(1996) 7:525-8; BioProbes Handbook, 2002 from Molecular Probes, Inc.;and Handbook of Fluorescent Probes and Research Products, Ninth Editionor Web Edition, from Molecular Probes, Inc, as well as the referencesherein. Many compounds, kits, etc. for use in caging various moleculesare commercially available, e.g., from Molecular Probes, Inc. Additionalreferences are found in, e.g., Merrifield, Science 232:341 (1986) andCorrie, J. E. T. and Trentham, D. R. (1993) In: Biological Applicationsof Photochemical Switches, ed., Morrison, H., John Wiley and Sons, Inc.New York, pp. 243-305. Examples of suitable photosensitive caging groupsinclude, but are not limited to, 2-nitrobenzyl, benzoin esters,N-acyl-7-nitindolines, meta-phenols, and phenacyls.

In some embodiments, a photoregulating (e.g., caging) group canoptionally comprise a first binding moiety, which can bind to a secondbinding moiety. For example, a commercially available cagedphosphoramidite[1-N-(4,4′-Dimethoxytrityl)-5-(6-biotinamidocaproamidomethyl)-1-(2-nitrophenyl)-ethyl]-2-cyanoethyl-(N,N-diisopropyl)-phosphoramidite(PC Biotin Phosphoramadite, from Glen Research Corp., www.glenres.com)comprises a photolabile group and a biotin (the first binding moiety). Asecond binding moiety, e.g., streptavidin or avidin, can thus be boundto the caging group, increasing its bulkiness and its effectiveness atcaging. In certain embodiments, a caged component comprises two or morecaging groups each comprising a first binding moiety, and the secondbinding moiety can bind two or more first binding moietiessimultaneously. For example, the caged component can comprise at leasttwo biotinylated caging groups; binding of streptavidin to multiplebiotin moieties on multiple caged component molecules links the cagedcomponents into a large network. Cleavage of the photolabile groupattaching the biotin to the component results in dissociation of thenetwork.

Traditional methods of creating caged polypeptides (including e.g.peptide substrates and proteins such as antibodies or transcriptionfactors) include, e.g., by reacting a polypeptide with a caging compoundor by incorporating a caged amino acid during synthesis of apolypeptide. See, e.g., U.S. Pat. No. 5,998,580 to Fay et al. (Dec. 7,1999) entitled “Photosensitive caged macromolecules”; Kossel et al.(2001) PNAS 98:14702-14707; Trends Plant Sci (1999) 4:330-334; PNAS(1998) 95:1568-1573; J. Am. Chem. Soc. (2002) 124:8220-8229;Pharmacology & Therapeutics (2001) 91:85-92; and Angew. Chem. Int. Ed.Engl. (2001) 40:3049-3051. A photolabile polypeptide linker (e.g., forconnecting a protein transduction domain and a sensor, or the like) can,for example, comprise a photolabile amino acid such as that described inU.S. Pat. No. 5,998,580.

Irradiation with light can, e.g., release a side chain residue of anamino acid that is important for activity of the peptide comprising suchamino acid. Additionally, in some embodiments, uncaged amino acids cancleave the peptide backbone of the peptide comprising the amino acid andcan thus, e.g., open a cyclic peptide to a linear peptide with differentbiological properties, etc.

Activation of a caged peptide can be done through destruction of aphotosensitive caging group on a photoregulated amino acid by anystandard method known to those skilled in the art. For example, aphotosensitive amino acid can be uncaged or activated by exposure to asuitable conventional light source, such as lasers (e.g., emitting inthe UV range or infrared range). Those of skill in the art will be awareof and familiar with a number of additional lasers of appropriatewavelengths and energies as well as appropriate application protocols(e.g., exposure duration, etc.) that are applicable to use withphotoregulated amino acids such as those utilized herein. Release ofphotoregulated caged amino acids allows control of the peptides thatcomprise such amino acids. Such control can be both in terms of locationand in terms of time. For example, focused laser exposure can uncageamino acids in one location, while not uncaging amino acids in otherlocations.

Those skilled in the art will appreciate a variety of assays can be usedfor evaluating the activity of a photoregulated amino acid, e.g., theassays described in the examples herein. A wide range of, e.g., cellularfunction, tissue function, etc. can be assayed before and after theintroduction of a peptide comprising a photoregulated amino acid intothe cell or tissue as well as after the release of the photoregulatedmolecule.

The compositions and methods herein can be utilized in a number ofaspects. For example, photoregulated amino acids (e.g., in peptides) candeliver therapeutic compositions to discrete locations of a body sincethe release or activation/deactivation/etc. of the photoregulated aminoacid can be localized through targeted light exposure, etc. It will alsobe appreciated that the methods, structures, and compositions of theinvention are applicable to incorporation/use of photoregulated naturalamino acids (e.g., ones with photoregulating moietiesattached/associated with them).

Photochromic and photocleavable groups can be used to spatially andtemporally control a variety of biological processes, either by directlyregulating the activity of enzymes (see, e.g., Westmark, et al., J. Am.Chem. Soc. 1993, 115:3416-19 and Hohsaka, et al., J. Am. Chem. Soc.1994, 116:413-4), receptors (see, e.g., Bartels, et al., Proc. Natl.Acad. Sci. USA, 1971, 68:1820-3; Lester, et al., Nature 1977, 266:373-4:Cruz, et al., J. Am. Chem. Soc., 2000, 122:8777-8; and, Pollitt, et al.,Angew. Chem. Int. Ed Engl., 1998, 37:2104-7), or ion channels (see,e.g., Lien, et al., J. Am. Chem. Soc. 1996, 118:12222-3; Borisenko, etal., J. Am. Chem. Soc. 2000, 122:6364-70; and, Banghart, et al., Nat.Neurosci. 2004, 7:1381-6), or by modulating the intracellularconcentrations of various signaling molecules (see, e.g., Adams, et al.,Annu. Rev. Physiol. 1993, 55:755-84). In general, this requires thechemical modification of either a protein or small molecule with aphotoreactive ligand such as azobenzene or a nitrobenzyl group. Theability to genetically incorporate photoresponsive amino acids intoproteins at defined sites directly in living organisms wouldsignificantly extend the scope of this technique. See, e.g., Wu, et al.,J. Am. Chem. Soc. 2004, 126:14306-7.

Kits

Kits are also a feature of the invention. For example, a kit forproducing a protein that comprises at least one unnatural amino acid ina cell is provided, where the kit includes a container containing apolynucleotide sequence encoding an O-tRNA, and/or an O-tRNA, and/or apolynucleotide sequence encoding an O-RS, and/or an O-RS. In oneembodiment, the kit further includes an unnatural amino acid such asp-ethylthiocarbonyl-L-phenylalanine, p-(3-oxobutanoyl)-L-phenylalanine,1,5-dansyl-alanine, 7-amino-coumarin alanine, 7-hydroxy-coumarinalanine, o-nitrobenzyl-serine, O-(2-nitrobenzyl)-L-tyrosine,p-carboxymethyl-L-phenylalanine, p-cyano-L-phenylalanine,m-cyano-L-phenylalanine, biphenylalanine, 3-amino-L-tyrosine,bipyridylalanine, p-(2-amino-1-hydroxyethyl)-L-phenylalanine;p-isopropylthiocarbonyl-L-phenylalanine; 3-nitro-L-tyrosine orp-nitro-L-phenylalanine. In another embodiment, the kit furthercomprises instructional materials for producing the protein.

EXAMPLES

The following examples are offered to illustrate, but not to limit theclaimed invention. One of skill will recognize a variety of non-criticalparameters that may be altered without departing from the scope of theclaimed invention.

Example 1 Orthogonal Translation Components for the In VivoIncorporation of 3-Nitro-L-Tyrosine into Proteins in E. coli

The present Example describes compositions and methods for thebiosynthetic incorporation of 3-nitro-L-tyrosine (see, FIG. 1) intoproteins using E. coli host cell translation machinery. Novel orthogonaltRNA/synthetase pairs derived from M. jannaschii were isolated thatfunction in an E. coli host cell system.

Novel orthogonal synthetases were derived from M. jannaschii tyrosyltRNA synthetase, and were used in conjunction with the previouslydescribed M. jannaschii suppressor tyrosyl-tRNA_(CUA) (SEQ ID NO: 1).These new orthogonal pairs have no affinity or very low affinity for anyof the common (i.e., naturally occurring) amino acids. The derivedorthogonal tRNA synthetases selectively charge the amber suppressortyrosyl-tRNA_(CUA) with 3-nitro-L-tyrosine. The aminoacylated suppressortRNA (i.e., the “charged” tRNA) is used as a substrate by endogenous E.coli translation apparatus to incorporate 3-nitro-L-tyrosine in responseto a TAG amber stop codon (a selector codon) encountered in atranscript. The orthogonality of these tRNA/synthetase pairs ensuresthat neither the tRNA nor the synthetases cross react with endogenous E.coli tRNAs or synthetases and that the unnatural amino acid getsdelivered only in response to TAG.

The novel synthetases were isolated using protocols previouslydescribed, see e.g., Alfonta et al., Journal of the American ChemicalSociety. 125(48):14662-14663 (2003); and International Publication WO2005/038002, published Apr. 28, 2005.

A library of M. jannaschii tyrosyl tRNA-synthetase mutants was generatedby mutagenesis of the wild-type M. jannaschii tyrosyl tRNA-synthetase.The amino acid and polynucleotide sequences of the wild-type M.jannaschii tyrosyl tRNA-synthetase molecule are shown in Table 5 andprovided in SEQ ID NOS: 3 and 4, respectively. The mutagenesis consistedof randomizing predicted active site residues based on the crystalstructure of the homologous tyrosyl tRNA-synthetase from Bacillusstearothermophilus.

Following mutagenesis, the pool of synthetases in the mutant library waspassed through five rounds of positive and negative selection. Thisselection yielded seven synthetase clones that had the ability to chargethe O-tRNA with 3-nitro-L-tyrosine, denoted clones A through G. Theseselected synthetase clones were sequenced, and their amino acidsequences were determined, as follows.

TABLE 2 Methanococcus jannaschii Amino acid position tyrosyl-tRNA SEQ IDsynthetase 32 67 70 155 158 167 NO: wild-type Tyr Ala His Gln Asp Ala 3clone A Val Val 7 Clone B Val Val 7 Clone C Val Val 7 Clone D Ser ThrAsn Thr 8 Clone E Val Val 7 Clone F Ala Pro Gly 9 Clone G Val Val 7

Clones A, B, C, E and G all converged to the same mutant sequence.Clones D and F showed different sequences. The amino acid sequences ofthese mutant synthetases are provided in Table 5, SEQ ID NOS: 7-9).

Example 2 Orthogonal Translation Components for the In VivoIncorporation of p-Nitro-L-Phenylalanine (NO₂-Phe) into Proteins in E.coli

This EXAMPLE describes the site-specific, genetically-programmedincorporation of p-nitro-L-phenylalanine (see, FIG. 1; also writtenNO₂-Phe) into proteins in E. coli using a novel orthogonal translationsystem.

The unnatural amino acid NO₂-Phe has been used as a photoaffinitylabeling probe to study protein-receptor structure (Dong, Mol.Pharmacol. 2005, 69, 1892), and as a fluorescence quencher toinvestigate protease activity (Wang, Biochem. Biophys. Res. Comm. 1994,201:835) and protein structure (Sisido, J. Am. Chem. Soc. 1998,120:7520; 2002, 124:14586). This amino acid has been incorporatedsite-specifically into proteins with an in vitro biosynthetic methodusing three-base (Schultz, Science 1989, 244:182), four-base (M. Sisido)and five-base (M. Sisido, Nucleic Acids Res. 2001,29, 3646) codons.However, this approach typically produces only small amounts of protein.Moreover, the method is limited due to the need of stoichiometricamounts of acylated tRNA and an inability to regenerate the aminoacyltRNA. In view of these limitations, we developed a novel in vivoorthogonal translation system to incorporate the unnatural amino aciddirectly into proteins, as described below.

To genetically encode NO₂-Phe in E. coli, the specificity of anorthogonal Methanococcus jannaschii tyrosyl-tRNA synthetase (MjTyrRS)was altered so that the synthetase specifically charges the mutanttyrosine amber suppressor tRNA (mutRNA_(CUA) ^(Tyr)) with the unnaturalamino acid NO₂-Phe. The mutant synthetase was derived from the screeningof a mutant MjTyrRS library. Positions for mutagenesis in that mutantlibrary were chosen in view of the analysis of the crystal structure ofa mutant MjTyrRS that selectively charges mutRNA_(CUA) ^(Tyr) withp-bromophenylalanine.

After several rounds of positive and negative selection usingmutRNA_(CUA) ^(Tyr) and the mutant MjTyrRS library in the presence orabsence of 1 mM of NO₂-Phe, respectively, a clone was evolved whosesurvival at high concentration of chloroamphenicol (90 μg/mL) wasdependent on the presence of NO₂-Phe. Moreover, green fluorescence wasonly observed for the selected clone in the presence of NO₂-Phe with aT7/GFPuv reporter with an amber selector codon at sites within thereporter gene. This result suggests that the evolved synthetase has ahigher specificity for NO₂-Phe than for any other natural amino acid.Sequencing of the clone revealed the following mutations in this evolvedsynthetase:

-   -   Tyr32→Leu    -   Glu 107→Ser    -   Asp158→Pro    -   Ile 159→Leu    -   His 160→Asn    -   Leu162→Glu        The nucleotide sequence of this clone is provided in Table 5,        SEQ ID NO: 11, and the corresponding amino acid sequence is        provided in Table 5, SEQ ID NO:10.

To test the ability of the evolved synthetase (mutNO₂-PheRS) andmutRNA_(CUA) ^(Tyr) to selectively incorporate NO₂-Phe into proteins, anamber stop codon was substituted at a permissive site (Lys7) in the genefor the Z domain protein with a C-terminal hexameric His tag. Cellstransformed with mutNO₂-PheRS, mutRNA_(CUA) ^(Tyr) and the Z domain genewere grown in the presence of 1 mM NO₂-Phe in GMML minimal media. Themutant protein was purified using an Ni²⁺ affinity column andsubsequently analysed by SDS-PAGE (see, FIG. 2) and MALDI-TOF (FIG. 3).The observed mass (m/e=7958) from MALDI-TOF analysis matches theexpected mass (m/e=7958) for the NO₂-Phe incorporated Z-domain protein.No Z domain was obtained in the absence of NO₂-Phe (see, FIG. 1),indicating a very high fidelity in the incorporation of the unnaturalamino acid.

Next, the feasibility of using the incorporated NO₂-Phe as afluorescence quencher was examined. From the reported fluorophorecounterparts of NO₂-Phe such as tyrosine, tryptophan, 1-pyrenylalanine,and β-anthraniloyl-l-α,β-diaminopropionic acid, the tryptophan/NO₂-Phepair was picked to incorporate into a model GCN4 leucine zipper, whichforms a parallel coiled-coil homodimer. The DNA binding region of theGCN4 gene (676-840 bp), which does not encode any tryptophan, was clonedfrom the yeast genome into the protein expression vector pET-26b.Subsequently, site-directed mutagenesis was utilized to substitute aminoacids in this protein at specific sites with either tryptophan or theNO₂-Phe unnatual amino acid (encoded by the TAG selector codon). TheGCN4 expression vector as well as a plasmid containing both mutNO₂-PheRSand mutRNA_(CUA) ^(Tyr) were cotransformed into E. coli BL21(DE3) cells,which were then grown in the presence of 1 mM NO₂-Phe in GMML minimalmedia. The accumulated GCN4pl mutant proteins were purified using anNi²⁺ affinity column and confirmed by SDS-PAGE and MALDI-TOF analyses.

Steady-state fluorescence spectra were measured for the purified mutantproteins. FIG. 4A shows the fluorescence spectrum of the ²²Trp mutantprotein alone and that of the mixture of ²²Trp and ²²NO₂-Phe mutants,while FIG. 4B shows the fluorescence spectrum of the ⁵⁵Trp mutantprotein and the spectrum of the mixture of ⁵⁵Trp and ²²NO₂-Phe mutants.A distinct fluorescence quenching was observed in 22Trp/22N₂-Phe mutantpair; on the other hand, no significant fluorescence quenching wasobtained for the ⁵⁵Trp/²²NO₂-Phe mutant pair. This result clearly showsthat the fluorophor/quencher interaction between Trp/NO₂-Phe pair isdistance-dependant. Thus, this system can readily be applied to thestudy of protein folding and protein-protein as well as protein-ligandinteractions.

Example 3 Orthogonal Translation Components for the In VivoIncorporation of the Redox Active Amino Acid 3-Amino-L-Tyrosine intoProteins in E. coli

The present Example describes compositions and methods for thebiosynthetic incorporation of 3-amino-L-tyrosine (see, FIG. 1; alsowritten NH₂—YRS) into proteins using E. coli host cell translationmachinery. Novel orthogonal synthetase/tRNA pairs derived from M.jannaschii for incorporating this unnatural amino acid were isolatedthat function in an E. coli host cell system.

This unnatural amino acid side chain is readily oxidized to thecorresponding semiquinone and quinone, thus can be used to both probeand manipulate electron transfer processes in proteins. The oxidizedquinone form can efficiently conjugate with acrylamide through ahetero-Diels-Alder reaction. This last property provides another use,namely where the unnatural amino acid serves as a handle for chemicalmodification of proteins.

Novel orthogonal synthetase were derived from M. jannaschii tyrosyl tRNAsynthetase, and were used in conjunction with the previously describedM. jannaschii suppressor tRNA_(CUA) (SEQ ID NO: 1) This new orthogonalpair has no affinity or very low affinity for any of the common (i.e.,naturally occurring) amino acids. The derived orthogonal tRNA synthetaseselectively charges the amber suppressor tRNA_(CUA) with3-amino-L-tyrosine. The aminoacylated suppressor tRNA (i.e., the“charged” tRNA) is used as a substrate by endogenous E. coli translationapparatus to incorporate 3-amino-L-tyrosine in response to the TAG amberstop codon (a selector codon) encountered in a transcript. Theorthogonality of these tRNA/synthetase pairs ensures that neither thetRNA nor the synthetases cross react with endogenous E. coli tRNAs orsynthetases and that the unnatural amino acid gets delivered only inresponse to TAG.

The novel synthetases were isolated using protocols that have beenpreviously described. A library of M. jannaschii tyrosyl tRNA-synthetasemutants was generated by mutagenesis of the wild-type M. jannaschiityrosyl tRNA-synthetase. The mutagenesis consisted of randomizingpredicted active site residues based on the crystal structure of otheraminoacyl tRNA-synthetase molecules.

Following mutagenesis, the pool of synthetases in the mutant library wassubjected to multiple rounds of positive and negative selection. Thisselection yielded one synthetase clone that had the ability to chargethe O-tRNA with 3-amino-L-tyrosine. This selected synthetase clone hasthe amino acid sequence shown in Table 5, SEQ ID NO: 12; and has thepolynucleotide sequence shown in Table 5, SEQ ID NO: 13.

Example 4 Orthogonal Translation Components for the In VivoIncorporation of the Phosphotyrosine Mimic Amino Acidp-Carboxymethyl-L-Phenylalanine into Proteins in E. coli

The present Example describes compositions and methods for thebiosynthetic incorporation of p-carboxymethyl-L-phenylalanine (see, FIG.1; also written pCMF) into proteins using E. coli host cell translationmachinery. Novel orthogonal synthetase/tRNA pairs derived from M.jannaschii for incorporating this unnatural amino acid were isolatedthat function in an E. coli host cell system.

This unnatural amino acid side chain can be used as a stable mimic fortyrosine phosphorylation. Tyrosine phosphorylation plays an importantrole in regulating cellular signal transduction in a broad range ofcellular processes, such as cell growth, metabolic regulation,transcriptional regulation, and proliferation. Tyrosine phosphorylationis a reversible process in vivo. The tendency to dephosphorylatetyrosine by endogenous tyrosine phosphatases interferes with studies ofthe effects of tyrosine phosphorylation, thus hindering theinterpretation of those studies. The amino acidp-carboxymethyl-L-phenylalanine is a phosphotyrosine mimic, is cellpermeable, and furthermore, does not serve as a substrate for tyrosinephosphatases. This unnatural amino acid, when incorporated intoproteins, can be used to generate protein mutants that areconstitutively active. This unnatural amino acid can also be used in thecontext of phage display to select for inhibitors to protein tyrosinephosphatase from libraries of peptides containingp-carboxymethyl-L-phenylalanine.

Novel orthogonal synthetases were derived from M. jannaschii tyrosyltRNA synthetase, and were used in conjunction with the previouslydescribed M. jannaschii suppressor tRNA_(CUA). These new orthogonalpairs have no affinity or very low affinity for any of the common (i.e.,naturally occurring) amino acids. The derived orthogonal tRNAsynthetases selectively charge the amber suppressor tRNA_(CUA) withp-carboxymethyl-L-phenylalanine. The aminoacylated suppressor tRNA(i.e., the “charged” tRNA) is used as a substrate by the endogenous E.coli translation apparatus to incorporatep-carboxymethyl-L-phenylalanine in response to a TAG amber stop codon (aselector codon) encountered in a transcript. The orthogonality of thesetRNA/synthetase pairs ensures that neither the tRNA nor the synthetasescross react with endogenous E. coli tRNAs or synthetases and that theunnatural amino acid gets delivered only in response to TAG.

A search for orthogonal synthetases that have the ability tospecifically charge an orthogonal tRNA withp-carboxymethyl-L-phenylalanine was undertaken. This search usedprotocols that have been previously described. A library of M.jannaschii tyrosyl tRNA-synthetase mutants was generated by mutagenesisof the wild-type M. jannaschii tyrosyl tRNA-synthetase, where themutagenesis consisted of randomizing predicted active site residuesbased on the crystal structure of other aminoacyl tRNA-synthetasemolecules.

Following mutagenesis, the mutant synthetase library was passed throughmultiple rounds of positive and negative selection. This selectionyielded five synthetase clones that had the ability to charge the O-tRNAwith p-carboxymethyl-L-phenylalanine. These synthetase clones weresequenced, and the amino acid sequences were determined, as shown inTable 5. The amino acid sequences of these O-RS clones is provided inSEQ ID NOS: 14, 16, 18, 20 and 22. The nucleotide sequences of thesesame O-RS clones is provided in SEQ ID NOS: 15, 17, 19, 21 and 23.

Example 5 Orthogonal Translation Components for In Vivo Incorporation ofthe Hydrophobic Unnatural Amino Acid Biphenylalanine into Proteins in E.coli

The present Example describes compositions and methods for thebiosynthetic incorporation of biphenylalanine (see, FIG. 1) intoproteins using E. coli host cell translation machinery. Novel orthogonalsynthetase/tRNA pairs derived from M. jannaschii for incorporating thisunnatural amino acid were isolated that function in an E. coli host cellsystem.

The biphenylalanine unnatural amino acid has a large aromatic sidechain. Hydrophobic interactions are one of the major forces that driveprotein folding and protein-protein interactions (the other major forcesare electrostatic interactions, hydrogen bonds, and van der waalsforces). Hydrophobic interactions are involved in many biologicalevents, such as protein transport across cell membranes, proteinaggregation, and enzyme catalysis. The hydrophobicity of biphenylalanineis higher than any of the common 20 amino acids. Incorporation ofbiphenylalanine into proteins is a useful tool in studying andmodulating intramolecular and intermolecular hydrophobic packinginteractions in proteins.

Novel orthogonal synthetases were derived from M. jannaschii tyrosyltRNA synthetase, and are used in conjunction with the previouslydescribed M. jannaschii suppressor tRNA_(CUA). Theses new orthogonalpairs have no affinity or very low affinity for any of the common (i.e.,naturally occurring) amino acids. The derived orthogonal tRNAsynthetases selectively charge the amber suppressor tRNA_(CUA) withbiphenylalanine. The aminoacylated suppressor tRNA (i.e., the “charged”tRNA) is used as a substrate by the endogenous E. coli translationapparatus to incorporate biphenylalanine in response to the TAG amberstop codon (a selector codon) encountered in a transcript. Theorthogonality of these tRNA/synthetase pairs ensures that neither thetRNA nor the synthetases cross react with endogenous E. coli tRNAs orsynthetases and that the unnatural amino acid gets delivered only inresponse to TAG.

A search for orthogonal synthetases that have the ability tospecifically charge an orthogonal tRNA with biphenylalanine wasundertaken. This search used protocols that have been previouslydescribed. A library of M. jannaschii tyrosyl tRNA-synthetase mutantswas generated by mutagenesis of the wild-type M. jannaschii tyrosyltRNA-synthetase, where the mutagenesis consisted of randomizingpredicted active site residues based on the crystal structure of otheraminoacyl tRNA-synthetase molecules.

Following mutagenesis, the mutant synthetase library was passed throughmultiple rounds of positive and negative selection. This selectionyielded seven synthetase clones that had the ability to charge theO-tRNA with biphenylalanine. These synthetase clones were sequenced, asshown in Table 5. The amino acid sequences of these O-RS clones isprovided in SEQ ID NOS: 24, 26, 28, 30, 32, 34 and 36. The correspondingnucleotide sequences of these same O-RS clones is provided in SEQ IDNOS: 25, 27, 29, 31, 33, 35 and 37.

Example 6 Orthogonal Translation Components for In Vivo Incorporation ofthe Metal-Chelating Unnatural Amino Acid Bipyridylalanine into Proteinsin E. coli

The present Example describes compositions and methods for thebiosynthetic incorporation of bipyridylalanine (see, FIG. 1) intoproteins using the E. coli host cell translation machinery. Novelorthogonal synthetase/tRNA pairs derived from M. jannaschii forincorporating this unnatural amino acid were isolated that function inan E. coli host cell system.

The bipyridylalanine unnatural amino acid has the ability to chelatemetal ions. The N,N-bidentate moiety of this amino acid side chain is astrong chelator to transition metal ions, such as Cu²⁺, Fe²⁺, Ni²⁺, Zn²⁺and Ru²⁺, etc. This metal chelating amino acid can be used to (1)introduce redox active or electrophilic metal ions into proteins, (2)form fluorescent metal ion complexes such as Ru(bpy)₃, or (3) mediatethe metal ion dependent dimerization of proteins containingbipyridylalanine.

Novel orthogonal synthetases were derived from M. jannaschii tyrosyltRNA synthetase, and were used in conjunction with the previouslydescribed M. jannaschii suppressor tRNA_(CUA). The new orthogonal pairshave no affinity or very low affinity for any of the common (i.e.,naturally occurring) amino acids. The derived orthogonal tRNA synthetaseselectively charged the amber suppressor tRNA_(CUA) withbipyridylalanine. The aminoacylated suppressor tRNA (i.e., the “charged”tRNA) is used as a substrate by the endogenous E. coli translationapparatus to incorporate bipyridylalanine in response to the TAG amberstop codon (a selector codon) encountered in a transcript. Theorthogonality of these tRNA/synthetase pairs ensures that neither thetRNA nor the synthetases cross react with endogenous E. coli tRNAs orsynthetases and that the unnatural amino acid gets delivered only inresponse to TAG.

A search for orthogonal synthetases that have the ability tospecifically charge an orthogonal tRNA with bipyridylalanine wasundertaken. This search used protocols that have been previouslydescribed. A library of M. jannaschii tyrosyl tRNA-synthetase mutantswas generated by mutagenesis of the wild-type M. jannaschii tyrosyltRNA-synthetase, where the mutagenesis consisted of randomizingpredicted active site residues based on the crystal structure of otheraminoacyl tRNA-synthetase molecules.

Following mutagenesis, the mutant synthetase library was passed throughmultiple rounds of positive and negative selection. This selectionyielded two synthetase clones that had the ability to charge the O-tRNAwith bipyridylalanine.

These synthetase clones were sequenced, as shown in Table 5. The aminoacid sequences of these O-RS clones is provided in SEQ ID NOS: 38 and40. The corresponding nucleotide sequences of these same O-RS clones isprovided in SEQ ID NOS: 39 and 41.

Example 7 Orthogonal Translation Components for In Vivo Incorporation ofthe Fluorescent Unnatural Amino Acid 1,5-Dansylalanine into Proteins inYeast Host Cells

The present Example describes compositions and methods for thebiosynthetic incorporation of 1,5-dansylalanine (see, FIG. 1) intoproteins using yeast host cell translation machinery. Novel orthogonalsynthetase/tRNA pairs derived from E. coli for incorporating thisunnatural amino acid were isolated that function in the yeast host cellsystem.

Fluorescence has become one of the most important detection signals inbiotechnology due to its high sensitivity and safety of handling.Moreover, processes like fluorescence resonance energy transfer (FRET)or fluorescence polarization make possible the real time analysis ofbiomolecular binding events, movements or conformational changes.Current fluorescent methodology to study proteins in vivo often rely onfusion constructs with large fluorescent proteins. Alternatively, smallorganic labels can be used to minimize structural perturbation, butexhibit poor regioselectivity, are cytotoxic or demand introduction ofdye binding protein motifs and are rather restricted to the proteinsurface. In contrast, a fluorescent amino acid does not necessarilycontain groups with cytotoxic potential, its introduction is only aminor alteration of protein structure and specific labeling is possibleat any position of the protein in vivo.

The present invention provides orthogonal translation system componentsthat incorporate the fluorescent amino acid 1,5-dansyl-modified alanine(see, FIG. 5A) in growing polypeptide chains in yeast. This unnaturalamino acid can also be identified by its IUPAC nomenclature:2-amino-3-(5-dimethylamino-naphthalene-1-sulfonylamino)-propionic acid.The dansyl chromophore has interesting spectral properties, including anexceptionally high separation of excitation and emission maxima (>200nm) and a high dependence of emission intensity on the polarity of theenvironment. This makes it well suited to study protein conformationalchanges or binding events where the local protein environment and thuspolarity is affected. Synthesis of the unnatural amino acid was achievedin a two-step procedure including coupling of N-Boc-aminoalanine todansylchloride using triethylamine in dichloromethane and subsequentacidic deprotection with TFA in dichloromethane.

Novel synthetases for incorporating 1,5-dansylalanine were isolatedusing protocols previously described, see e.g., Wu et al., Journal ofthe American Chemical Society 126:14306-14307 (2004); and InternationalApplication No. PCT/US 2005/034002, filed Sep. 21, 2005, by Deiters etal. A mutant E. coli leucyl-tRNA synthetase clone (clone B8) thatdisplayed initial charging activity was isolated from a randomized E.coli leucyl-tRNA synthetase library in a yeast host cell system. See,Table 5 and SEQ ID NOS: 42 and 43. The sites in the mutant E. colileucyl-tRNA synthetase library were M40, L41, Y499, Y527 and H537.Additional mutations (caused during library construction) found in allclones throughout the library were H67R, N196T, R262A and S497C.

However, the B8 mutant E. coli synthetase exhibited background activitytowards one or more natural amino acids with a weight similar to leucineas judged by MALDI TOF MS of the expressed model protein humansuperoxide dismutase bearing a permissive amber codon at position 33(hSOD-33TAG-His6). Theoretical docking studies with dansylalanine-AMPamide and a crystal structure of the homologous leucyl-tRNA synthetasefrom Thermus thermophilus (T. th.) suggested formation of an enlargedbinding pocket that binds the ligand by mainly hydrophobic interactionswithout participation of π-stacking to the naphtyl moiety (see, FIG.5B).

A proofreading activity is present in E. coli leucyl-tRNA synthetase,and since activating and charging activity towards 1,5-dansylalanine wasalready evolved in the selected mutant, a strategy was devised thattargeted the selective removal of activated or charged natural aminoacids by remodelling the editing site. It was contemplated that theobserved background was due to incorporation of leucine as suggested byMALDI TOF MS. Crystal structures of the homologous leucyl-tRNAsynthetase from T. th. and mutational studies suggest that a simplesteric block of unpolar amino acids towards the γ-methyl side chainprevents activated or charged leucine from binding to the hydrolyticsite (Lincecum et al., Mol. Cell., 4:951-963 [2003]).

To increase hydrolytic activity towards leucine, residues T252 and V338in the E. coli synthetase editing domain were exchanged to alanine byQuikchange mutagenesis in order to enlarge the binding pocket (see, FIG.6A). The V338A synthetase (see, Table 5, SEQ ID NOS: 46 and 47) did notexhibit significant difference in expression studies using the modelprotein human superoxide dismutase (hSOD), whereas the T252A synthetase(see, Table 5, SEQ ID NOS: 44 and 45) showed a marked reduction inbackground (see, FIG. 6B). High selectivity of this mutant was furtherconfirmed by MALDI TOF MS of hSOD-33TAG-His6.

Thus, the invention provides novel mutant tRNA-synthetases derived fromE. coli leucyl-tRNA synthetase that have the ability to biosyntheticallyincorporate 1,5-dansylalanine into proteins using yeast (e.g.,Saccharomyces cerevisiae) host cell translation machinery.

Example 8 Orthogonal Translation Components for In Vivo Incorporation ofthe Photocaged Unnatural Amino Acid o-Nitrobenzylserine into Proteins inYeast Host Cells

The present Example describes compositions and methods for thebiosynthetic incorporation of o-nitrobenzylserine (see, FIG. 1) intoproteins using yeast host cell translation machinery. Novel orthogonalsynthetase/tRNA pairs derived from E. coli for incorporating thisunnatural amino acid were isolated that function in the yeast host cellsystem.

The investigation of function of a specific gene in living organismsmostly relies on its deactivation or activation and studying of theresulting effects. Classic genetic knockout studies target the gene onthe DNA level, leading to inactivation of the production of all encodedprotein variants and do not allow real time investigation of resultingeffects. In recent years, the use of small organic molecules hasdramatically increased the specificity of gene deactivation. Using suchtools, a single protein variant (or a single domain of that variant) canbe targeted and effects can be investigated in real time after additionof the molecule.

The introduction of photocaged amino acids into proteins as transient,activatable knockouts can further increase the accuracy of such studies.Using chemical knockout strategies, diffusion time of the compound toits target protein can be rate limiting and it is only possible toinvestigate a whole cell. In contrast, photouncaging of specific aminoacids can be performed on a rapid timescale and specific compartments ofa cell can be investigated using pulsed and highly focused laser light.

A mutant E. coli leucyl-tRNA synthetase has previously been evolved froma mutant E. coli leucyl-tRNA synthetase library in yeast host cells thatspecifically recognizes the caged cysteine derivativeo-nitrobenzylcysteine, also written o-NBC (see, Wu et al., Journal ofthe American Chemical Society 126:14306-14307 (2004); and InternationalApplication No. PCT/US 2005/034002, filed Sep. 21, 2005, by Deiters etal.). To expand the applicability of this approach, the evolution of anaminoacyl-tRNA synthetase specifically incorporating o-nitrobenzylserine(oNBS) was contemplated. When genetically incorporated into proteins,this photocaged unnatural amino acid could be used to photoregulate anyfunction involving serine residues, for example but not limited to,serine phosphorylation by kinases, representing one of the mostimportant chemical markers in signal transduction pathways. The oNBSamino acid can be synthesized by coupling o-nitrobenzylic bromide toBoc-N-Ser-O-tBu in DMF using NaH as base and subsequent acidicdeprotection with TFA in methylenechloride under presence oftriethylsilane as scavenger with 52% overall yield.

The mutant E. coli leucyl-tRNA synthetase evolved for oNBC incorporation(clone 3H11; see Table 5, SEQ ID NOS: 48 and 49) already exhibited somelimited activity for incorporating oNBS, but with about twofold reducedefficiency compared to an oNBC amino acid. To evolve a more efficientoNBS translation system, the selected clone 3H11 synthetase wasdiversified by error prone PCR, again using three differentmutagenicities, to introduce one, two or five mutations per gene,yielding an overall diversity of 1×10⁷ clones. The positions in theleucyl-tRNA-synthetase enzyme that were targeted for randomization wereM40, L41, Y499, Y527 and H537. The protocols used herein follow thegeneral methodologies described in the art, e.g., Wu et al., Journal ofthe American Chemical Society 126:14306-14307 (2004); and InternationalApplication No. PCT/US 2005/034002, filed Sep. 21, 2005, by Deiters etal.

Screening of the new mutant synthetase library yielded an improvedsynthetase (clone G2-6) with twofold enhanced oNBS incorporationefficiency. Sequencing of the G2-6 clone identified five additionalmutations throughout the enzyme in comparison to the initial 3H11synthetase starting material (positions S31G, T247A, T248S, M617I andV673A). Additional mutations (caused during library construction) foundin all clones throughout the library were also observed as follows:H67R, N196T, R262A and S497C. The complete amino acid and nucleotidesequences of this synthetase isolate are provided in Table 5, SEQ IDNOs: 50 and 51. This improved mutant synthetase is illustratedschematically in FIG. 7A, and the improvement in oNBS incorporationactivity in the G2-6 synthetase mutant is illustrated experimentally inFIG. 7B. The selective incorporation of oNBS was further confirmed byMALDI MS again using hSOD as model system for expression studies.

Thus, the invention provides a novel mutant tRNA-synthetase derived fromE. coli leucyl-tRNA synthetase that has the ability to biosyntheticallyincorporate oNBS into proteins using yeast (e.g., Saccharomycescerevisiae) host cell translation machinery.

Example 9 Orthogonal Translation Components for In Vivo Incorporation ofthe Photocaged Unnatural Amino Acid O-(2-Nitrobenzyl)-L-Tyrosine intoProteins in E. coli

The present Example describes compositions and methods for thebiosynthetic incorporation of O-(2-nitrobenzyl)-L-tyrosine (see, FIG. 1)into proteins using Archae synthetase species and E. coli host celltranslation machinery. Novel orthogonal synthetase/tRNA pairs derivedfrom M. jannaschii for incorporating this unnatural amino acid wereisolated that function in the E. coli host cell system.

“Caged proteins” are modified proteins whose biological activity can becontrolled by light, usually by photolytic conversion from an inactiveto an active form. This is particularly useful since irradiation can beeasily controlled in timing, location and amplitude, enabling detailedstudies of protein function (for reviews, see Shigeri et al., Pharmacol.Therapeut. 2001, 91:85; Curley and Lawrence Pharmacol. Therapeut. 1999,82:347; Curley and Lawrence, Curr. Op. Chem. Bio. 1999, 3:84; “CagedCompounds”, Methods in Enzymology; Marriott, G., Ed.; Academic Press:New York, 1998; V. 291; and Adams and Tsien, Annu. Rev. Physiol. 1993,55:755).

The most common caging groups are 2-nitrobenzyl groups (see, Bochet, J.Chem. Soc., Perkin 12002, 125; Givens et al., Methods in Enzymology1998, 291, 1; and Pillai, Synthesis 1980, 1), which are installed onhydroxy, carboxy, thio, or amino groups of polypeptides or proteins andare readily cleaved upon irradiation with non-photodamaging UV light.Previously, caged proteins were produced by chemical modification ofisolated proteins without positional control on the caging groupinstallation and also mostly resulting in the incorporation of multiplecaging groups (e.g., Self and Thompson, Nature Med. 1996, 2, 817). Otherexamples employ the in vitro incorporation of a caged amino acid using anonsense codon suppression technique (see, Philipson et al., Am. J.Physiol. Cell. Physiol. 2001, 281, C195; Pollitt and Schultz Angew.Chem. Int. Ed. 1998, 37, 2105; Cook et al., Angew. Chem. Int. Ed. 1995,34, 1629). Since the aminoacylated-tRNA has to be synthesizedchemically, only small quantities of protein are accessible and in vivostudies are limited.

The use of orthogonal translation system technology has overcome theinherent limitations in these technologies. Using cellular systems,non-natural amino acids can be site-specifically incorporated with hightranslational fidelity into proteins in vivo by addition of newcomponents to the translational machinery of E. coli (for review, see,for example, Wang and Schultz, Angew. Chem. Int. Ed. 2004, 44, 34; Croppand Schultz, Trend. Gen. 2004, 20, 625; and Wang and Schultz, Chem.Commun. 2002, 1).

The present Example describes the addition of a photocaged tyrosine,O-(2-nitrobenzyl)-L-tyrosine (see FIG. 1), to the genetic code of E.coli. Tyrosine is an important amino acid in protein tyrosine kinase andphosphatase substrates, it is an essential residue in several enzymeactive sites, and it is often located at protein-protein interfaces.

Irradiation of O-(2-nitrobenzyl)-L-tyrosine (synthesized from L-tyrosineas described in Miller et al., Neuron 1998, 20, 619) at 365 nm inducescleavage of the benzylic CO-bond and rapid formation of the decagedamino acid (t_(1/2)=4 min, see supporting information), as illustratedschematically in FIG. 8.

Photodecaging of O-(2-nitrobenzyl)-L-tyrosine can be experimentallyobserved, as illustrated in the experimental results shown in FIG. 9. Asshown in this figure, the photodecaging of O-(2-nitrobenzyl)-L-tyrosinewas studied by irradiation of a 0.2 mM solution in water (one well of asix-well plate) using a handheld UV lamp (365 nm at 10 mm distance).Aliquots were taken at specific time points and analyzed by LC/MS. Theconcentrations of O-(2-nitrobenzyl)-L-tyrosine (squares) and the decagedspecies (circles) are shown in the figure. 50% decaging was achievedafter approximately four minutes.

The Methanococcus jannaschii tyrosyl tRNA-synthetase (MjYRS) was used asa starting point for the generation of an orthogonal synthetase thataccepts O-(2-nitrobenzyl)-L-tyrosine, but not any of the 20 common aminoacids as a substrate. MjYRS does not aminoacylate any endogenous E. colitRNAs with tyrosine, but aminoacylates a mutant tyrosine ambersuppressor (mutRNA_(CUA)). To alter the specificity of the MjYRS toselectively recognize O-(2-nitrobenzyl)-L-tyrosine, a library ofapproximately 10⁹ YRS mutants was generated by randomizing six residues(Tyr32, Leu65, Phe108, Gln109, Asp158 and Leu162) in the tyrosinebinding pocket, based on the crystal structure of the M. jannaschiiYRS/tRNA^(Tyr)-tyrosine complex (Zhang et al., Prot. Sci. 2005, 14,1340; Kobayashi et al., Nat. Struct. Biol. 2003, 10, 425). These sixresidues were chosen based on their close proximity to the para positionof the phenyl ring of tyrosine, among which Tyr32 and Asp158 formhydrogen bonds with the hydroxyl group of tyrosine. Mutations of theseresidues are expected to expand the substrate binding pocket of thesynthetase to specifically recognize O-(2-nitrobenzyl)-L-tyrosine andother unnatural amino acids.

Active synthetase variants were screened from the mutant MjYRS libraryusing chloramphenicol acetyl transferase (CAT) and barnase reportersystems for positive and negative selections, respectively. After fiverounds of alternating positive and negative selection, 96 clones werescreened for a phenotype in the presence and absence ofO-(2-nitrobenzyl)-L-tyrosine. Three synthetases were furthercharacterized using an in vivo assay based on suppression of theAsp112TAG codon in the CAT gene. E. coli expressing the threeMjYRS/mutRNA_(CUA) pairs survived on chloramphenicol with IC₅₀ values of110 mg/L and less than 10 mg/L in the presence and absence ofO-(2-nitrobenzyl)-L-tyrosine (1 mM), respectively. The large differencein chloramphenicol resistance suggests a substantial in vivo specificityof the selected synthetase/tRNA pairs for insertion ofO-(2-nitrobenzyl)-L-tyrosine over all 20 natural amino acids in responseto an amber codon.

Nucleic acids encoding these three O-(2-nitrobenzyl)-L-tyrosine-tRNAsynthetases were sequenced, and their amino acid sequences were deduced.The complete amino acid sequences of the three ONBY synthetase clones isprovided in Table 5, SEQ ID NOs: 52-54. The results of this sequencingare shown in Table 3.

TABLE 3 synthetase Amino Acid Position species (mutant codon) MjYRS 3265 108 109 158 162 wild-type RS Tyr Leu Phe Gln Asp Leu ONBY RS-1 GlyGly Ala Arg Glu Tyr (GGG) (GGT) (GCG) (CGT) (GAG) (TAT) ONBY RS-2 AlaGly Cys Asp Ala Gly (GCT) (GGG) (TGT) (GAT) (GCG) (GGT) ONBY RS-3 GlyGly Glu Gln Ser Glu (GGG) (GGT) (GAG) (CAG) (TCG) (GAG)

Conceivably, the mutations Tyr32→Gly32/Ala32 and Asp158→Glu158, Ala158,or Ser158 result in the loss of hydrogen bonds between Tyr32, Asp158,and the natural substrate tyrosine, thus disfavoring its binding.

To measure the fidelity and efficiency of the three ONB-MjYRSs,O-(2-nitrobenzyl)-L-tyrosine was incorporated in response to an ambercodon at position four in a C-terminally hexahistidine tagged mutantsperm whale myoglobin gene. To express recombinant protein, plasmidpBAD/JYAMB-4TAG (which encodes the mutant sperm whale myoglobin genewith an arabinose promoter and an rrnB terminator; the tyrosyltRNA_(CUA) on an lpp promoter and an rrnC terminator; and a tetracyclineresistance marker) was co-transformed with a pBK vector (encoding themutant synthetase and a kanamycin resistance gene) into DH10B E. coli inthe presence of both the synthetase/mutRNA_(CUA) pair andO-(2-nitrobenzyl)-L-tyrosine (1 mM). Cells were amplified inLuria-Bertani media (5 mL) supplemented with tetracycline (25 mg/L) andkanamycin (30 mg/L), washed with phosphate buffer, and used toinnoculate 500 mL of liquid glycerol minimal media (GMML; glycerolminimal media supplemented with 0.3 mM leucine) containing theappropriate antibiotics, photocaged tyrosine (1 mM), and arabinose(0.002%). Cells were grown to saturation and then harvested bycentrifugation.

Purified mutant myoglobin protein was obtained by Ni-NTA affinitychromatography with a yield of approximately 2-3 mg/L and judged tobe >90% homogeneous by SDS-PAGE and Gelcode Blue staining. The yield iscomparable to myoglobin expression using the wild typeMjYRS/mutRNA_(CUA) pair suppressing the same amber codon. No myoglobinwas detectable if the unnatural amino acid was withheld or in thepresence of 1 mM tyrosine, revealing a very high selectivity of allthree synthetases for O-(2-nitrobenzyl)-L-tyrosine (see, FIG. 10).

To further confirm the identity of the site-specifically photocagedprotein, a different myoglobin mutant with an amber codon at Gly74 (dueto superior mass spectrometry properties) was expressed in the presenceof pONB-MjYRS-1, tRNA_(CUA), and O-(2-nitrobenzyl)-L-tyrosine (1 mM).The myoglobin mutant 74TAG was expressed, under the same conditions asthe 4TAG mutant, using the synthetase pONB-1 in presence ofO-(2-nitrobenzyl)-L-tyrosine (1 mM) and purified by nickel affinitycolumn. Protein bands were visualized by Gelcode Blue staining of anSDS-PAGE gel and excised from the polyacrylamide gel. The gel pieceswere sliced into 1.5-mm cubes and subjected to trypsin hydrolysisessentially as described (Shevchenko et al., Anal. Chem. 1996, 68,850-858). Tryptic peptides were analyzed by liquid chromatography tandemmass spectrometry (LC-MS/MS) analysis performed on a Finnigan LCQ Decaion trap mass spectrometer (Thermo Finnigan) fitted with a NanosprayHPLC (Agilent 1100 series). The precursor ions corresponding to thesingly and doubly charged ions of the peptide HGVTVLTALGJILK containingthe unnatural amino acid (denoted J) were separated and fragmented withan ion trap mass spectrometer. The LC-MS/MS analysis shows Tyrosine atposition 74 (tryptic peptide HGVTVLTALGYILK). The fragment ion massescould be assigned, indicating the site-specific incorporation oftyrosine (3) at position 74 (see, FIGS. 11A and 11B). The detection ofTyr74 is most likely due to a fragmentation of the labile benzylether inO-(2-nitrobenzyl)-L-tyrosine during MS analysis.

To confirm the previous incorporation of the caged amino acidO-(2-nitrobenzyl)-L-tyrosine, the deuterated derivative was synthesizedand used in an expression of the same myoglobin mutant under identicalconditions. The protein was then trypsinized and subjected to ananalysis by mass spectrometry. An assignment of the fragment ion massesrevealed d₂-tyrosine incorporation at position 74 of myoglobin,unambiguously demonstrating the incorporation of the unnatural aminoacid 2 (see FIGS. 12A and 12B). The LC MS/MS analysis did not indicateincorporation of any natural amino acid at this position, providingadditional evidence for the high fidelity of the evolved synthetase.

Additionally, the in vivo photochemical activation of a protein havingO-(2-nitrobenzyl)-L-tyrosine incorporated can be demonstrated byemploying lacZ as a reporter gene. E. coli β-galactosidase displays anessential tyrosine at position 503 (Juers et al., Biochemistry 2001, 40,14781; Penner et al., Biochem. Cell Biol. 1999, 77, 229). Thecorresponding codon was mutated to an amber stop codon TAG for theincorporation of the caged O-(2-nitrobenzyl)-L-tyrosine. The3-galactosidase is monitored before and after tyrosine decaging. Theβ-galactosidase activity is restored following irradiation in vivo.

Example 10 Orthogonal Translation Components for In Vivo Incorporationof the Unnatural Amino Acid p-Cyanophenylalanine into Proteins in E.coli

The present Example describes compositions and methods for thebiosynthetic incorporation of p-cyanophenylalanine (see, FIG. 1; alsowritten 4-cyanophenylalanine) into proteins using E. coli host celltranslation machinery. Novel orthogonal synthetase/tRNA pairs derivedfrom M. jannaschii for incorporating this unnatural amino acid wereisolated that function in an E. coli host cell system.

The cyano group is an excellent local environment IR probe, as its CNstretching vibration (ν₂) undergoes a frequency shift on the order often wave numbers when moved from hydrophobic to hydrophilic surroundings(Getahun et al., “Using Nitrile-Derivatized Amino Acids as InfraredProbes of Local Environment,” JACS 125, 405-411 [2003]). Para(4-position) and meta (3-position) cyanophenylalanine are thus useful instudying an assortment of protein properties including protein-proteinbinding, protein conformation, and hydrophobic collapse.

Para and meta forms of cyanophenylalanine can exist in both polar andhydrophobic environments while in a peptide chain, and their effects onconformation are negligible. Thus, either is likely to reside in thesame environment as the wild-type residue it replaces in a protein orpeptide. Further, the compounds' CN stretching vibration is narrow, doesnot overlap with any other protein absorptions, is largely decoupledfrom the protein's other vibrations, and is quite sensitive to changesin solvent polarity. For these reasons, both are excellent tools forpeptide conformational studies.

Aromatic Nitriles as Local Environment IR Probes in Small Peptides

Getahun and coworkers (Getahun et al., “Using Nitrile-Derivatized AminoAcids as Infrared Probes of Local Environment,” JACS 125, 405-411[2003]) have shown (see, FIG. 13) that the cyano stretching vibration ofpara-cyanophenylalanine is ten wavenumbers higher in water than in THF.When the 14-residue amphipathic peptide mastoparan x (MPx) is mutated toincorporate para-cyanophenylalanine into its lipid binding portion, thecyano stretch is at 2229.6 cm⁻¹ when MPx is bound to a POPC lipidbilayer. In water, the MPx PheCN mutant's CN stretching vibration occursat 2235.7 cm⁻¹. In sum, the cyano stretch in a hydrated peptide issimilar to the free para-cyanophenylalanine cyano stretch in water,while the PheCN cyano stretch in a buried peptide is similar to the freePheCN cyano stretch TI-IF (Tucker et al., “A New Method for Determiningthe Local Environment and Orientation of Individual Side Chains ofMembrane-Binding Peptides,” JACS 126 5078-5079 [2004]).

A novel orthogonal synthetase was derived from M. jannaschii tyrosyltRNA synthetase, and are used in conjunction with the previouslydescribed M. jannaschii suppressor tRNA_(CUA). This new orthogonal pairhas no affinity or very low affinity for any of the common (i.e.,naturally occurring) amino acids. The derived orthogonal tRNA synthetaseselectively charges the amber suppressor tRNA_(CUA) withp-cyanophenylalanine. The aminoacylated suppressor tRNA (i.e., the“charged” tRNA) is used as a substrate by the endogenous E. colitranslation apparatus to incorporate p-cyanophenylalanine in response tothe TAG amber stop codon (a selector codon) encountered in a transcript.The orthogonality of these tRNA/synthetase pairs ensures that neitherthe tRNA nor the synthetases cross react with endogenous E. coli tRNAsor synthetases and that the unnatural amino acid gets delivered only inresponse to TAG.

A search for orthogonal synthetases that have the ability tospecifically charge an orthogonal tRNA with p-cyanophenylalanine wasundertaken. This search used protocols that have been previouslydescribed. A library of M. jannaschii tyrosyl tRNA-synthetase mutantswas generated by mutagenesis of the wild-type M. jannaschii tyrosyltRNA-synthetase, where the mutagenesis consisted of randomizingpredicted active site residues based on the crystal structure of otheraminoacyl tRNA-synthetase molecules.

Following mutagenesis, the mutant synthetase library was passed throughmultiple rounds of positive and negative selection. This selectionyielded one synthetase clone that had the ability to charge the O-tRNAwith p-cyanophenylalanine. This synthetase clone was sequenced, and theamino acid sequence was determined, as shown in Table 5, SEQ ID NOs: 55and 56). This synthetase mutant shows the follow substitutions relativeto the wild-type synthetase sequence: Tyr32Leu, Leu65Val, Phe108Trp,Gln109Met, Asp158Gly and Ile159Ala.

Example 11 Orthogonal Translation Components for in vivo Incorporationof the Unnatural Amino Acid m-Cyanophenylalanine into Proteins in E.coli

The present Example describes compositions and methods for thebiosynthetic incorporation of m-cyanophenylalanine (see, FIG. 1; alsowritten 3-cyanophenylalanine) into proteins using E. coli host celltranslation machinery. Novel orthogonal synthetase/tRNA pairs derivedfrom M. jannaschii for incorporating this unnatural amino acid wereisolated that function in an E. coli host cell system.

The cyano group is an excellent local environment IR probe, as its CNstretching vibration (ν₂) undergoes a frequency shift on the order often wave numbers when moved from hydrophobic to hydrophilic surroundings(Getahun et al., “Using Nitrile-Derivatized Amino Acids as InfraredProbes of Local Environment,” JACS 125, 405-411 [2003]). Para(4-position) and meta (3-position) cyanophenylalanine are thus useful instudying an assortment of protein properties including protein-proteinbinding, protein conformation, and hydrophobic collapse.

Para and meta forms of cyanophenylalanine can exist in both polar andhydrophobic environments while in a peptide chain, and their effects onconformation are negligible. Thus, either is likely to reside in thesame environment as the wild-type residue it replaces in a protein orpeptide. Further, the compounds' CN stretching vibration is narrow, doesnot overlap with any other protein absorptions, is largely decoupledfrom the protein's other vibrations, and is quite sensitive to changesin solvent polarity. For these reasons, both are excellent tools forpeptide conformational studies.

Aromatic Nitriles in Proteins

Following established directed evolution protocols, a novelMethanococcus jannaschii tRNATyrCUA-tyrosyl-tRNA synthetase (TyrRS) pairwas evolved that site specifically incorporates meta-cyanophenylalaninewith high fidelity in response to an amber TAG codon. This neworthogonal pair has no affinity or very low affinity for any of thecommon (i.e., naturally occurring) amino acids. The derived orthogonaltRNA synthetase selectively charges the amber suppressor tRNA_(CUA) withm-cyanophenylalanine. The aminoacylated suppressor tRNA (i.e., the“charged” tRNA) is used as a substrate by endogenous E. coli translationapparatus to incorporate m-cyanophenylalanine in response to the TAGamber stop codon (a selector codon) encountered in a transcript. Theorthogonality of these tRNA/synthetase pairs ensures that neither thetRNA nor the synthetases cross react with endogenous E. coli tRNAs orsynthetases and that the unnatural amino acid gets delivered only inresponse to an amber nonsense codon, TAG.

Construction of the orthogonal synthetase that has the ability tospecifically charge an orthogonal tRNA with m-cyanophenylalanine usedprotocols that have been previously described. A library of M.jannaschii tyrosyl tRNA-synthetase mutants was generated by mutagenesisof the wild-type M. jannaschii tyrosyl tRNA-synthetase, where themutagenesis consisted of randomizing predicted active site residuesbased on the crystal structure of other aminoacyl tRNA-synthetasemolecules.

Following mutagenesis, the mutant synthetase library was passed throughmultiple rounds of positive and negative selection. This selectionyielded a synthetase clone that had the ability to charge the O-tRNAwith m-cyanophenylalanine. This synthetase clone was sequenced, and theamino acid sequence was determined (see, Table 5, SEQ ID NOs: 57 and58). This synthetase mutant shows the follow substitutions relative tothe wild-type synthetase sequence: Tyr32His, His70Ser, Asp158Ser,Ile159Ser and Leu162Pro.

We attempted to suppress a Tyr7→TAG mutant of the c-terminal His₆-taggedZ-domain protein in both the presence and absence ofm-cyanophenylalanine and p-cyanophenylalanine, using their respectiveorthogonal tRNA/synthetase pair. In both cases, full length protein wasproduced in the presence of unnatural amino acid, while no product wasdetectable by Coomasssie blue staining on an SDS-PAGE gel in the absenceof the respective unnatural amino acid.

Further, we have obtained IR spectra of this protein with both meta andpara-cyanophenylalanine incorporated into position 7. After backgroundsubtraction of the wild-type z-domain IR spectrum, we obtained thespectra shown in FIGS. 14A and 14B.

FIG. 14A shows that para-cyanophenylalanine has a single absorbancebetween the extremes shown in FIG. 13, suggesting that residue numberseven lies along the surface of the protein, but does not point directlyinto solution. FIG. 14B shows the spectrum of meta-cyanophenylalanine asthe sum of two Gaussian distributions with peaks at 2236 and 2228 cm⁻¹.The R² value for the curves is greater than 0.99, an excellentcurve-fit, and data in FIG. 14B thus suggest that m-cyanophenylalaninehas two conformations. As evidenced by the peak at 2228 cm⁻¹, oneconformation places the cyano group in a hydrophobic region of theprotein. The peak at 2236 cm⁻¹ suggests that the other conformationplaces it in a hydrated environment.

Example 12 Orthogonal Translation Components for in vivo Incorporationof the Unnatural Amino Acid p-(2-Amino-1-Hydroxyethyl)-L-Phenylalanineinto Proteins in E. coli

The present Example describes compositions and methods for thebiosynthetic incorporation of p-(2-amino-1-hydroxyethyl)-L-phenylalanine(see, FIG. 1) into proteins using E. coli host cell translationmachinery. Novel orthogonal synthetase/tRNA pairs derived from M.jannaschii for incorporating this unnatural amino acid were isolatedthat function in an E. coli host cell system.

The site-specific modification of proteins with biophysical probes,cytotoxic agents, cross-linking agents, and other agents has been widelyused to analyze protein structure and function, and in the developmentof diagnostics, therapeutic agents, and high-throughput screening. Oneapproach to the selective modification of proteins involves theoxidation of an N-terminal serine or threonine to the correspondingaldehyde and subsequent coupling with hydrazine, alkoxyamine, orhydrazide derivatives. Unfortunately, this method is limited since itcan only be used to modify the N-terminal position of a protein. Ourapproach of placing the aminoalcohol critical functional group of2-amino-1-hydroxyethyl onto a target protein's side chain will removethe limitation of selective protein modification on the N-terminus onlywith the added benefit of controlling the position of the aminoalcoholgroup in protein.

A novel orthogonal synthetase was derived from M. jannaschii tyrosyltRNA synthetase, and is used in conjunction with the previouslydescribed M. jannaschii suppressor tRNA_(CUA). This new orthogonal pairhas no affinity or very low affinity for any of the common (i.e.,naturally occurring) amino acids. The derived orthogonal tRNA synthetaseselectively charges the amber suppressor tRNA_(CUA) withp-(2-amino-1-hydroxyethyl)-L-phenylalanine. The aminoacylated suppressortRNA (i.e., the “charged” tRNA) is used as a substrate by endogenous E.coli translation apparatus to incorporatep-(2-amino-1-hydroxyethyl)-L-phenylalanine in response to the TAG amberstop codon (a selector codon) encountered in a transcript. Theorthogonality of this tRNA/synthetase pair ensures that neither the tRNAnor the synthetase cross reacts with endogenous E. coli tRNAs orsynthetases and that the unnatural amino acid gets incorporated only inresponse to an amber nonsense codon, TAG.

A search for orthogonal synthetases that have the ability tospecifically charge an orthogonal tRNA withp-(2-amino-1-hydroxyethyl)-L-phenylalanine was undertaken. This searchused protocols that have been previously described. A library of M.jannaschii tyrosyl tRNA-synthetase mutants was generated by mutagenesisof the wild-type M. jannaschii tyrosyl tRNA-synthetase, where themutagenesis consisted of randomizing predicted active site residuesbased on the crystal structure of other aminoacyl tRNA-synthetasemolecules.

Following mutagenesis, the mutant synthetase library was passed throughmultiple rounds of positive and negative selection. This selectionyielded one synthetase clone that had the ability to charge the O-tRNAwith p-(2-amino-1-hydroxyethyl)-L-phenylalanine. That synthetase clonewas sequenced, and the amino acid sequence was determined (see, Table 5,SEQ ID NO: 59). This synthetase mutant shows the follow substitutionsrelative to the wild-type M. janaschii synthetase sequence:

wild-type M. janaschii  Tyr Leu Phe Gln Asp Leu tyrosyl-tRNA synthetase32 65 108 109 158 162 mutant synthetase specific Asp Glu Arg Gln Gly Asnfor p-(2-amino-1- (GAT) (GAG) (CGT) (CAG) (GGG) (AAT)hydroxyethyl)-L-phenylalanine (mutant codon)

Example 13 Orthogonal Translation Components for in vivo Incorporationof the Unnatural Amino Acid p-Ethylthiocarbonyl-L-Phenylalanine intoProteins in E. coli

The present Example describes compositions and methods for thebiosynthetic incorporation of p-ethylthiocarbonyl-L-phenylalanine (see,FIG. 1) into proteins using E. coli host cell translation machinery.Novel orthogonal synthetase/tRNA pairs derived from M. jannaschii forincorporating this unnatural amino acid were isolated that function inan E. coli host cell system.

A useful method for the generation of semisynthetic proteins is nativechemical ligation in which two fully unprotected peptide fragments canbe coupled by an amide bond under mild physiological conditions at roomtemperature (Nilsson et al., Annu. Rev. Biophys. Biomol. Struct. 2005,34, 91-118; Dawson et al., Science 1994, 266, 776-779). A variation ofthis method, termed expressed protein ligation in which one or bothreaction partners have been produced by recombinant means, is useful forthe synthesis of proteins consisting of greater than 100 residues (Muir,Annu. Rev. Biochem 2003, 72, 249-289; David et al. Eur. J. Biochem.2004, 271, 663-677). In practice, both techniques require the presenceof a C-terminal α-thioester, limiting these methods to modification atthe C-terminus of a peptide fragment. The placement of a reactivethioester group at any residue in a bacterially expressedpeptide/protein would significantly expand the scope of thesetechniques, allowing, for example, the synthesis of cyclic or branchedstructures or the selective modification of side chains with biophysicalprobes, polyethylene glycols or various tags. Methods for the generationof proteins having thioester-containing side chains find use in that thethioester-containing side chains can participate in subsequent chemicalligation reactions in vitro and possibly in vivo (Camarero and Muir, J.Am. Chem. Soc. 1999, 121, 5597-5598; Camarero et al., Bioorg Med. Chem.2001, 9, 2479-2484; Scott et al., Proc. Natl. Acad. Sci. USA 1999, 96,13638-13643; Evans et al., J. Biol. Chem. 2000, 275, 9091-9094; Yeo etal., Chem. Commun. 2003, 2870-2871).

Synthesis of P-Ethylthiocarbonyl-L-Phenylalanine

p-ethylthiocarbonyl-L-phenylalanine (structure 1; also termed4-(ethylthiocarbonyl)-L-phenylalanine) was synthesized in four steps(see, FIG. 15) starting from commercially available α-bromo-p-toluicacid (1a) and N-(diphenylmethylene)glycine tert-butyl ester (1c). Thesesteps are outlined below.

Synthesis of S-ethyl 4-(bromomethyl)benzothioate (structure 1b)

To a solution of 1a (2.15 g, 10.0 mmol) in THF (50 ml) was added thionylchloride (2 ml, 28 mmol), followed by addition of DMF (50 μl) and thereaction mixture was stirred for 5 hours at room temperature. Theorganic solvents were removed under reduced pressure until a white solidappeared, which was then dissolved in THF (50 ml) and the solution wascooled to 0° C. A solution of ethanethiol (0.78 ml, 10.0 mmol) andtriethylamine (2 ml, 14 mmol) in THF (10 ml) was added dropwise over acourse of 30 minutes. The reaction mixture was stirred for another fourhours and solvent was removed. Water (100 ml) and ether (200 ml) wereadded. The organic phase was washed with H₂O (2×50 mL), dried overNaSO₄, and removed under reduced pressure. The crude product waspurified by flash chromatography on silica gel (8% ethyl acetate inhexane), yielding 1b (2.18 g, 78%) as a colorless oil. ¹H NMR (400 MHz,CDCl₃) δ 7.94 (d, J=8.0 Hz, 2 H), 7.46 (d, J=8.0 Hz, 2H), 4.50 (s, 2 H),3.07 (q, J=7.6, 15.2 Hz, 2 H), 1.35 (t, J=9.2 Hz, 3 H). Exact mass m/zcalculated for C₁₀H₁₁BrOS 258.0/260.0. found (LC/MS) 259.1/260.1.

Synthesis of tert-butyl2-(diphenylmethyleneamino)-3-(4-(ethylthiocarbonyl) phenyl) propanoate(structure 1d)

A solution containing 1b (0.455 g, 1.76 mmol), 1c (0.47 g, 1.60 mmol),18-crown-6 (0.42 g, 1.59 mmol) and anhydrous K₂CO₃ (0.344 g, 2.50 mmol)in anhydrous CH₃CN (10 ml) was stirred for 24 hours at room temperature.The organic solvents were removed under reduced pressure. Water (100 ml)and CH₂Cl₂ (200 ml) were added. The organic phase was washed with H₂O(2×50 mL), dried over NaSO₄, and removed under reduced pressure. Thecrude product was used directly in the next step without purification.Exact mass m/z calculated for C₂₉H₃₁NO₃S 473.2. found (LC/MS) 474.3.

Synthesis of (4-(ethylthiocarbonyl))phenylalanine (1)

A solution of 1d (0.94 g, 2.0 mmol) from the previous step intrifluoroacetic acid (8 ml) and CH₂Cl₂ (2 ml) was stirred for one hourat room temperature. After the organic solvents were completely removedunder reduced pressure, concentrated HCl solution (0.8 ml) and MeOH (10ml) were added and the resulting solution was stirred for 1 hour at roomtemperature, after which time all solvent was removed and anhydrousacetone (10 ml) was added. The solution was filtered and the recoveredsolid was trituated with anhydrous MeOH (2 ml). After filtration, themethanolic filtrate was subjected to reduced pressure to afford 1 as awhite solid (>0.55 g, 95%). ¹H NMR (400 MHz, DMSO-d6) δ 7.85 (d, J=8.0Hz, 2 H), 7.44 (d, J=8.0 Hz, 2 H), 4.21 (t, J=6.4 Hz, 1 H), 3.06 (q,J=14.8, 17.2 Hz, 2 H), 3.00 (s, 2 H), 1.26 (t, J=7.2 Hz, 3 H). Exactmass m/z calculated for C₁₂H₁₅NO₃S 253.1. found (LC/MS) 254.2.

Genetic Programming of P-Ethylthiocarbonyl-L-Phenylalanine Incorporation

To genetically encode p-ethylthiocarbonyl-L-phenylalanine in E. coli, itwas necessary to generate an orthogonal aminoacyl-tRNA synthetase/tRNApair specific for this amino acid. On the basis of a crystal structureof the M. jannaschii TyrRS-tRNA L-tyrosine complex (Kobayashi et al.,Nat. Struct. Biol. 2003, 10, 425-432), six residues (Tyr³², Leu⁶⁵,Phe¹⁰⁸, Gln¹⁰⁹, Asp¹⁵⁸ and Leu¹⁶²) in the tyrosine-binding site of M.jannaschii TyrRS were randomly mutated. A library of 10⁹ TyrRS mutantswas passed through three rounds of positive selection (based on thesuppression of an amber codon in chloramphenicol acetyltransferase)alternated with two rounds of negative selection (based on suppressionof three amber codons in the barnase gene) in the presence and absenceof p-ethylthiocarbonyl-L-phenylalanine, respectively, and a number ofclones emerged whose survival in chloramphenicol was dependent onp-ethylthiocarbonyl-L-phenylalanine. One of these mutants was found tosupport cell growth in 120 μg mL⁻¹ chloramphenicol in the presence ofp-ethylthiocarbonyl-L-phenylalanine, and 10 μg mL⁻¹ chloramphenicol inits absence.

Sequencing of this clone revealed the following mutations: Tyr32Ala,Leu65Phe, Phe108Trp, Gln109Ser, Asp158Ser and Leu162H is (see, Table 5,SEQ ID NO: 60). The mutation of Tyr³² to Ala³² likely removes thehydrogen bond between the phenolic hydroxyl group of bound tyrosine andTyr³².

wild-type M. janaschii Tyr Leu Phe Gln Asp Leu tyrosyl-tRNA synthetase32  65 108 109 158 162 mutant synthetase specific Ala Phe Trp Ser SerHis for p-ethylthiocarbonyl-L- (GCT) (TTT) (TGG) (AGT) (TCG) (CAT)phenylalanine (mutant codon)

To confirm that the observed phenotype is caused by the site-specificincorporation of p-ethylthiocarbonyl-L-phenylalanine by themutRNA_(CUA)-mutTyrRS pair, an amber codon was substituted for theseventh position (Tyr) in the gene encoding the Z domain protein(Nilsson et al., Protein Eng. 1987, 1, 107-113) fused to a C-terminalHis₆ tag. Protein was expressed in the presence or absence of 1 mMp-ethylthiocarbonyl-L-phenylalanine and purified by Ni-NTAchromatography. Analysis by SDS-PAGE showed that expression of themutant Z domain protein was completely dependent on the presence ofp-ethylthiocarbonyl-L-phenylalanine. The mutant protein was expressed inapproximately 10-30% yield relative to the wide-type Z domain protein(−8 mg/L in minimal medium containing 1% glycerol, 0.3 mM leucine and 1mM p-ethylthiocarbonyl-L-phenylalanine with appropriate antibiotics).

Additional evidence for the site specific incorporation ofp-ethylthiocarbonyl-L-phenylalanine was obtained by matrix-assistedlaser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). In addition to the observation of an experimental average mass of8006 Da for the intact Tyr p-ethylthiocarbonyl-L-phenylalanine protein(M_(Theoretical)=8002 Da, see, FIG. 16), a minor peak corresponding tothe mutant protein without the first methionine moiety in acetylatedform (M_(Experimental)=7913 Da vs. M_(Theoretical)=7913 Da) and a majorpeak corresponding to the mutant protein without the first methioninemoiety (M_(Experimental)=7871 Da vs. M_(Theoretical)=7871 Da) were alsodetected (FIG. 16). Another major peak of 7828 Da was present thatcorresponds to Z domain protein containing a free carboxylic acid groupinstead of a thioester moiety at position 7, which has a calculated massof 7827 Da in its protonated form. With the assumption that both acidand thioester-containing mutant proteins have comparable ionizationefficiencies under mass detection conditions, the integration of theircorresponding mass peak areas suggests that around 40%thioester-containing mutant protein is hydrolyzed. The fact that themutant synthetase does not incorporate unnatural amino acidp-ethylthiocarbonyl-L-phenylalanine in its hydrolysized form in vivo andthat p-ethylthiocarbonyl-L-phenylalanine appears to be stable both invitro and in vivo, suggest that the hydrolysis of the thioester into theacid occurs after its incorporation into Z domain protein by thethioester-specific mutant synthetase.

To determine whether the thioester side chain of the mutant proteins canbe selectively modified, an in vitro chemical ligation was performedwith 20-60 μg/ml crude thioester-containing mutant protein and 10 mMcysteine ethyl ester in a phosphate buffered solution containing 100 mMdithiothreitol (DTT) and 2 M guanidinium chloride at a pH 8.0. Theresulting modified protein was then purified and analyzed by MALDI-TOFMS. Experimental average masses of 7956 Da and 7998 Da, corresponding tothioester-containing proteins modified with one cysteine molecule(M_(Theoretical)=7958 Da and 8000 Da for proteins without the firstmethionine moiety and without the first methionine moiety in acetylatedform, respectively), were obtained (FIG. 17). The labeling efficiencycould be qualitatively estimated to be greater than 85% by theintegration of their mass peak areas.

As shown in FIG. 16, Z domain proteins are predominantly expressedwithout the first methionine residue. In this form, unmodifiedthioester-containing mutant protein has a molecular weight of 7872,which may overlap with the acid-containing proteins in acetylated form(M_(Theoretical)=7869 Da). Therefore, in calculating the labelingefficiency, the peak area at 7867 Da was taken as the upper-limit valuefor unmodified thioester-containing proteins, leading to an estimate ofgreater than 85% labeling efficiency.

The peaks at 7825 Da and 7867 Da (M_(Theoretical)=7827 Da and 7869 Da)are indicative of the presence of Z domain proteins containing acarboxylic acid group at position 7, which is not reactive towardcysteine ethyl ester. As expected, no labeling products were detectedfor WT Z domain proteins, indicating that the labeling reaction occurredonly between the cysteine molecule and the thioester group but not anyexisting functional groups in the WT protein. On the other hand, neitherintramolecular side chain cyclization nor self-dimerization involvingthe thioester group and any ε-amino group of the five lysine residues inthioester-containing mutant proteins were observed. These data,therefore, demonstrate the excellent selectivity and reactivity of thethioester handle for the reliable and selective in vitro modification ofproteins.

Chemical Ligation Between Cysteine Ethyl Ester and Thioester-ContainingZ Domain Proteins

E. coli DH10B cells (60 ml) harboring plasmid encoding the mutant tRNAsynthetase and expression vector pLEIZ encoding Z domain gene with anamber codon at the 7^(th) position and a COOH-terminal His-6 tag weregrown at 37° C., induced for four hours at an OD₆₀₀ of 0.5 by theaddition of 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) andpelleted. To the pelleted cells was added 1 ml buffer solution (6Mguanidinium chloride, 100 mM sodium phosphate, 200 mM sodium chloride,pH=8.0). The solution was shaken for 1 hour at room temperature,sonicated for three minutes and centrifuged to remove any cell debris.To the clear supernatant was added 2 ml of phosphate buffered solution(100 mM sodium phosphate, 200 mM sodium chloride, 0.01M cysteine ethylester, ph=8.0), 150 μl dithiothreitol solution (2 M) and 60 mg of sodium2-mercaptoethenesulfonate (MESNA). The solution mixture was shaken for12 hours at room temperature. The solution containing modified proteinswas exchanged and concentrated into 500 μl buffer solution (8 M urea,100 mM sodium phosphate, 10 mM Trizma, pH 8.0). Modified proteins werepurified by Ni²⁺ affinity chromatography according to manufacturer'sprotocol (Qiagen, Chatsworth, Calif.), dialyzed against distilled waterand analyzed by MALDI-TOF MS.

Conclusion

In conclusion, we have provided a novel orthogonal synthetase derivedfrom M. jannaschii tyrosyl tRNA synthetase. When used in conjunctionwith an M. jannaschii suppressor tRNA_(CUA), these reagents allow the invivo incorporation of the unnatural amino acidp-ethylthiocarbonyl-L-phenylalanine in polypeptide chains. This workillustrates a biosynthetic protocol for bacterial production of proteinscontaining a side chain thioester handle at defined sites. This allowsthe highly selective and efficient chemical ligation of a wide varietyof ligands to the reactive group on thep-ethylthiocarbonyl-L-phenylalanine amino acid residue followingincorporation of the amino acid into a protein.

Example 14 Orthogonal Translation Components for in vivo Incorporationof the Diketone Unnatural Amino Acid p-(3-Oxobutanoyl)-L-Phenylalanineinto Proteins in E. coli

The present Example describes compositions and methods for thebiosynthetic incorporation of the diketone unnatural amino acidp-(3-oxobutanoyl)-L-phenylalanine (see, FIG. 1) into proteins using E.coli host cell translation machinery. Novel orthogonal synthetase/tRNApairs derived from M. jannaschii for incorporating this unnatural aminoacid were isolated that function in an E. coli host cell system.

The comparative studies on the ability of simple monoketone orβ-diketone functional groups to form imines with butylamine and thestabilities of thus formed imines in phosphate buffer at different pHsdemonstrate the facile production of enol imine formed from theβ-diketone moiety at pHs ranging from 6.5 to 10.5, as well as itssuperior stability toward the acidic hydrolysis down to pH 3.9. Incomparison, at a pH up to 10.5 under the identical conditions, themonoketone group essentially remains as a free form with no detectableimine formation. Accordingly, an unnatural amino acid bearing aβ-diketone moiety on its side chain was synthesized and theidentification of an orthogonal tRNA-synthetase pair capable ofincorporating this unnatural amino acid was undertaken. The inventionprovides a successfully evolved mutant synthetase that specificallyincorporation of this diketone-containing amino acid into proteins invivo with high translational efficiency and fidelity. As described morefully below, a biotin hydroxylamine derivative was then selectivelycoupled to this diketone group that was genetically encoded into a Zdomain protein, suggesting that the diketone moiety could serve as apowerful chemical handle whose reactivity is orthogonal to normalbiological chemistries for bringing a variety of external propertiesinto the target proteins.

It has been previously demonstrated that by adding new components to thetranslational machinery of Escherichia coli or yeast, noncanonical aminoacids could be site-specifically incorporated with high translationalfidelity and efficiency into proteins in vitro or in vivo usingorthogonal translation components. This approach has been used togenetically incorporate ketone-containing amino acids into proteins,which could subsequently be conjugated with nonpeptidic molecules withdiverse biological and/or physical properties (e.g., polyethyleneglycol, biotin, glycomimetics, etc) through the formation of hydrazoneand oxime bonds. Although these hydrazone and oxime bonds are stable atthe physiological conditions, they are disadvantaged by the requirementfor the simultaneous presence of two functional groups that are notfound among the common twenty amino acids. If one had a reactivefunctional group such as a thioester or β-diketone that directly formedstable adducts with ε-amino group of lysines or α-amino groups, onecould form intermolecular or intramolecular protein crosslinks. To thisend, we now report the genetic encoding of the diketone-containing aminoacid 2 in Escherichia coli. See, FIG. 21.

It was contemplated that the conjugated product of an aryl diketone 2with an aliphatic amine may lead to the formation of imine adducts 3(see, FIG. 21), which can tautomerize to the corresponding enaminesstabilized by a six-membered intramolecular hydrogen bond. This mayresult in a stable adduct at physiological pH. To experimentally verifythis rationale, we began by measuring the relative reactivity of asimple model system that includes a series of imine formations betweenbutylamine and the aryl monoketone 1a and the aryl diketone 2 in 100 mMphosphate buffer at different pHs ranging from 6.5 to 10.5. The variousadducts (1b and 3a-3d) were assayed using liquid chromatography massspectrometry (LC/MS). See, Table 4. This Table describes the results ofthe imine formation between butylamine (10 mM) and either arylmonoketone 1 (1 mM) or 2 (1 mM) in PBS buffer (100 mM K(PO₄)_(i), 500 mMNaCl) at different pHs. Reactions were conducted for one week at roomtemperature.

TABLE 4 Conversion percentage to their respective imine adducts atdifferent pHs 75% MeOH Substrate pH 6.5 pH 7.4 pH 8.4 pH 9.5 pH 10.5 inH2O** 1 0% 0% 0% 0% 0% >90% 2 30% 50% 50% 57% 75% >90% **12 hoursreaction time at room temperatureThis analysis showed that, at pH up to 10.5, 1a essentially remains as afree form with no detectable formation of 1b. In contrast, at a pH of7.4, 50% of 2 have already been converted into 3 and this percentageincreases to an impressive value of 75% at pH 10.5.

This, taken together with the previous observations that keto form 2band enol-imine form 3b dominates over other corresponding taumers inaqueous medium (Iglesias, Curr. Org. Chem. 2004, 8, 1-24; Patteux etal., Org. Lett. 2003, 5, 3061-3063; Aly, Tetrahedron 2003, 50,1739-1747; Lopez et al., Tetrahedron: Asymmetry 1998, 9, 3741-3744;Mazzone et al., S. Eur. J. Med. Chem. 1986, 21, 277-284; and Kim andRyu, Bull. Korean. Chem. Soc. 1992, 13, 184-187), thereby confirmingthat the hydrogen-bonding induced stabilization does significantlyfacilitate the production of 3 (predominantly 3b) when compared to 1a.

To further corroborate that the stabilized intramolecular H-bond renders3 a greater stability toward the hydrolysis than simple imine 1b, LC/MSanalysis were also performed on both 1b and 3 at pH ranging from 1.9 to9.4. As demonstrated in FIG. 1, 3 (presumably 3b) essentially remainintact at the physiological pH of 7.4 or above. Only ˜40% conversion of3 to 2 occurs at a pH down to 3.9 after 4 days at room temperature. Amore acidic treatment of 3 led to the complete uninstallation of theamino group (FIG. 18). In sharp contrast but as expected, 1b is readilyhydrolyzed even at a pH 10.5 after overnight stirring (data not shown).

Unnatural Amino Acid Synthesis

Encouraged by these findings, we wished to establish the chemistry forderivatizing an unnatural amino acid p-(3-oxobutanoyl)-L-phenylalaninecontaining a β-diketone moiety in its side chain. Our synthesis strategy(see, FIG. 19) starts from readily accessiblep-acetyl-(±)-L-phenylalanine by protecting the backbone amino and acidgroups by Boc chemistry and esterification, respectively. The additionof a second carbonyl group was accomplished under reaction conditionsinvolving potassium tert-butoxide in a mixed solvent (2:3 [v/v] methylacetate:THF). The removal of Boc group with TFA, followed by alkalinehydrolysis, delivered the desired p-(3-oxobutanoyl)-L-phenylalanine withan overall yield of 40%.

Identification of Orthogonal Translation Components

Novel orthogonal aminoacyl-tRNA synthetase/tRNA pairs were constructedfor in vivo incorporation of p-(3-oxobutanoyl)-L-phenylalanine intoproteins using established protocols. On the basis of the crystalstructure of the M. jannaschii TyrRS-tRNA(Tyr) L-tyrosine complex(Kobayashi et al., Nat. Struct. Biol. 2003, 10, 425-432), six residues(Tyr³², Leu⁶⁵, Phe¹⁰⁸, Gln¹⁰⁹, Asp¹⁵⁸ and Leu¹⁶²) around thetyrosine-binding site of M. jannaschii TyrRS were randomly mutated.After sequentially passing the generated library of approximately 10⁹mutants through three rounds of positive selection, alternated with tworounds of negative selection according to our published protocol, anumber of clones emerged whose survival in chloramphenicol was dependenton the presence of p-(3-oxobutanoyl)-L-phenylalanine. Two TyrRS mutantswere identified by using an in vivo assay based on the suppression ofthe Asp¹¹² TAG codon in the CAT gene. These two mutants can support cellgrowth in 120 μg mL⁻¹ chloramphenicol in the presence ofp-(3-oxobutanoyl)-L-phenylalanine, and up to 10 μg mL⁻¹ chloramphenicolwithout p-(3-oxobutanoyl)-L-phenylalanine. This result suggests that thetwo evolved synthetases both have higher activity forp-(3-oxobutanoyl)-L-phenylalanine than for any natural amino acid.Sequencing the DNA of these mutants revealed that they converged to thesame sequence (see, Table 5, SEQ ID NO: 61).

Both hydrogen bonds between the phenolic hydroxy group of bound tyrosineand Tyr³² and Asp¹⁵⁸ are disrupted by mutations to Gly. Leu⁶⁵ isconverted to Val⁶⁵, possibly providing more space to accommodate theextended backbone of the β-diketone. The mutations of Phe108Thr andLeu162Ser as well as a conserved Gln¹⁰⁹ may thus indicate theirinvolvement in H-bonding to the carbonyl oxygen in β-diketone moiety.The sequences of these synthetase clones is summarized below.

wild-type M. janaschii Tyr Leu Phe Gln Asp Leu tyrosyl-tRNA synthetase32 65 108 109 158 162 mutant synthetase specific Gly Val Thr Gln Gly Serfor p-(3-oxobutanoyl)-L- (GGT) (GTT) (ACT) (CAG) (GGG) (AGT)phenylalanine (mutant codon)

To confirm that the observed phenotype is caused by the site-specificincorporation of p-(3-oxobutanoyl)-L-phenylalanine by the mutRNA_(CUA)^(Tyr)-mutTyrRS pair, an amber codon was introduced in place of thecodon for tyrosine at the seventh position in the gene encoding the Zdomain protein (Nilsson et al., Protein Eng. 1987, 1, 107-113) fused toa C-terminal His₆ tag. Protein was expressed in the presence or absenceof 1 mM p-(3-oxobutanoyl)-L-phenylalanine and purified by Ni-NTAchromatography. Analysis by SDS-PAGE revealed unnatural amino aciddependent protein expression (FIG. 20). The volume of mutant proteinloaded into the gel is three times the wide type (WT) protein wherediketone-containing unnatural amino acid is replaced with a tyrosineresidue, indicating around 30% incorporation efficiency ofp-(3-oxobutanoyl)-L-phenylalanine compared to tyrosine.

More convincing evidence for the unambiguous incorporation ofp-(3-oxobutanoyl)-L-phenylalanine was obtained by matrix-assisted laserdesorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS).In addition to the observation of an experimental average mass of 7991Da (M_(Theoretical)=7997 Da) for the intact protein, a major peakcorresponding to the protein without the first methionine moiety(M_(Experimental)=7867 Da, M_(Theoretical)=7866 Da) was also detected.The signal-to-noise ratio was >400, suggesting a fidelity for theincorporation of p-(3-oxobutanoyl)-L-phenylalanine of better than 99%using evolved mutRNA_(CUA) ^(Tyr)-mutTyrRS pair.

The possibility of using a diketone moiety as a chemical handle forsite-specific modification of protein with external properties wastested by carrying out in vitro labeling of expresseddiketone-containing proteins with biotin hydroxylamine derivative(MW=331.39, purchased from Molecular Probes). The purified mutant and WTZ domain proteins were treated with 2 mM biotin hydroxylamine inphosphate buffer at a pH 4.0 at 25° C. for 12 hours. After dialysisagainst water to remove excess biotin hydroxylamine, the proteins wereanalyzed by MALDI-TOF MS. Experimental average masses of 8315 Da(M_(Theoretical)=8310 Da, biotin-labeled intact mutant protein), 8182 Da(M_(Theoretical)=8179 Da, biotin-labeled mutant protein without thefirst methionine residue), and 8225 Da (M_(Theoretical)=8221 Da,biotin-labeled mutant protein without the first methionine residue inits acetylated form) were obtained, confirming that biotin hydroxylaminereacted with the mutant Z domain proteins in a molar ratio of 1:1. Asexpected, no labeling products were detected for WT Z domain proteins,indicating that the labeling reaction occurred only between thehydroxylamine and the diketone group, but not any existing functionalgroups in the WT protein. Taken together with no observation ofunlabeled diketone-containing mutant proteins in the mass spectrum,these data demonstrate the excellent specificity and high reactivity ofthe diketone handle for the selective in vitro modification of proteins.

The present Example demonstrates that the incorporation of a β-diketonehandle into protein in vivo using an evolved and highly specificorthogonal translation system occurs site specifically with a highfidelity and efficiency that is comparable with its natural counterpart.Given both the high stability and facile production of 3 over a broad pHrange, the modulation of protein-protein interactions through theformation of Schiff base between the β-diketone moiety and the aminogroup of a lysine residue is highly possible, especially whenp-(3-oxobutanoyl)-L-phenylalanine is placed in a favorable hydrophobicenvironment.

Example 15 Orthogonal Translation Components for in vivo Incorporationof the Unnatural Amino Acid p-Isopropylthiocarbonyl-L-Phenylalanine intoProteins in E. coli

The present Example describes compositions and methods for thebiosynthetic incorporation of p-i sopropylthiocarbonyl-L-phenylalanine(see, FIG. 1) into proteins using E. coli host cell translationmachinery. Novel orthogonal synthetase/tRNA pairs derived from M.jannaschii for incorporating this unnatural amino acid were isolatedthat function in an E. coli host cell system. This unnatural amino acidfinds use as a target for port-translational modifications whenincorporated into proteins, and is further advantageous because thechemically reactive moiety on the unnatural amino acid is resistant tothe hydrolysis activities of cellular enzymes.

A novel orthogonal synthetase was derived from M. jannaschii tyrosyltRNA synthetase, and is used in conjunction with the previouslydescribed M. jannaschii suppressor tRNA_(CUA). This new orthogonal pairhas no affinity or very low affinity for any of the common (i.e.,naturally occurring) amino acids. The derived orthogonal tRNA synthetaseselectively charges the amber suppressor tRNA_(CUA) withp-isopropylthiocarbonyl-L-phenylalanine. The aminoacylated suppressortRNA (i.e., the “charged” tRNA) is used as a substrate by endogenous E.coli translation apparatus to incorporatep-isopropylthiocarbonyl-L-phenylalanine in response to the TAG amberstop codon (a selector codon) encountered in a transcript. Theorthogonality of this tRNA/synthetase pair ensures that neither the tRNAnor the synthetase cross reacts with endogenous E. coli tRNAs orsynthetases and that the unnatural amino acid gets incorporated only inresponse to an amber nonsense codon, TAG.

A search for orthogonal synthetases that have the ability tospecifically charge an orthogonal tRNA withp-isopropylthiocarbonyl-L-phenylalanine was undertaken. This search usedprotocols that have been previously described. A library of M.jannaschii tyrosyl tRNA-synthetase mutants was generated by mutagenesisof the wild-type M. jannaschii tyrosyl tRNA-synthetase, where themutagenesis consisted of randomizing predicted active site residuesbased on the crystal structure of other aminoacyl tRNA-synthetasemolecules.

Following mutagenesis, the mutant synthetase library was passed throughmultiple rounds of positive and negative selection. This selectionyielded one synthetase clone that had the ability to charge the O-tRNAwith p-isopropylthiocarbonyl-L-phenylalanine. That synthetase clone wassequenced, and the amino acid sequence was determined (see, Table 5, SEQID NO: 62). This synthetase mutant shows the follow substitutionsrelative to the wild-type M. janaschii synthetase sequence:

wild-type M. janaschii Tyr Leu Phe Gln Asp Leu tyrosyl-tRNA synthetase32 65 108 109 158 162 mutant synthetase specific Gly Cys Cys Met Gly Tyrfor p-isopropylthiocarbonyl- GGG) (TGT) (TGT) (ATG) (GGT) (TAT)L-phenylalanine (mutant codon)

Example 16 Orthogonal Translation Components for in vivo Incorporationof Fluorescent Unnatural Amino Acids Containing Coumarin into Proteinsin E. coli

The present Example describes compositions and methods for thebiosynthetic incorporation of 7-amino-coumarin alanine and7-hydroxy-coumarin alanine (see, FIG. 1) into proteins using E. colihost cell translation machinery. Novel orthogonal synthetase/tRNA pairsderived from M. jannaschii for incorporating this unnatural amino acidwere isolated that function in an E. coli host cell system.

Fluorescence is one of the most sensitive and useful techniques inmolecular biology. The discovery of Green Fluorescent Protein (GFP) hasled to a dramatic revolution in cell biology, allowing the study ofprotein expression, localization, dynamics and interaction in livingcells by direct visualization (Lippincott-Schwartz et al., Nat. Rev.Mol. Cell. Bio. (2001) 2:444-456). However, the protein interaction anddynamics cannot be pinpointed at atomic resolution due to the size ofGFP. GFP also requires many transcripts to achieve a suitable signal,and required a lag-time for its folding and fluorophore maturation.

The incorporation of fluorescent amino acids, as opposed to an entirefluorescent protein moiety, would overcome some of the limitations inthe GFP fluorescence system. The site-specific incorporation offluorescent amino acids would introduce minimum perturbation to the hostprotein, which permits the measurement of fluorescence resonance energytransfer (FRET) with much greater precision (Truong and Ikura, Curr.Opin. Struct. Bio. 2001, 11:573-578). In addition, the use of afluorescent amino acid will permit the probing of the local environmentof each amino acid position, and pinpoint the residues that mediateinteraction with other cellular components by varying the position ofthe fluorescent amino acid in the protein. This would also be veryuseful to study protein folding in vitro (Lakowicz, J. R. Principles ofFluorescence Spectroscopy Ed. 2; Kluwer Academic/Plenum Publishers: NewYork, 1999), especially in a single-molecular system (Lipman et al.,Science 2003, 301:1233-1235), because one protein molecule normallycontains more than one tryptophan residue, and specific chemicallabeling of proteins with fluorescent probes is extremely difficult.

The coumarin alanines shown in FIG. 1 have been chemically synthesized.A novel orthogonal synthetase was derived from M. jannaschii tyrosyltRNA synthetase, and is used in conjunction with the previouslydescribed M. jannaschii suppressor tRNA_(CUA) to incorporate thesecoumarin amino acids. This new orthogonal pair has no affinity or verylow affinity for any of the common (i.e., naturally occurring) aminoacids. The derived orthogonal tRNA synthetase selectively charges theamber suppressor tRNA_(CUA) with 7-amino-coumarin alanine and7-hydroxy-coumarin alanine. The aminoacylated suppressor tRNA (i.e., the“charged” tRNA) is used as a substrate by the endogenous E. colitranslation apparatus to incorporate 7-amino-coumarin alanine and7-hydroxy-coumarin alanine in response to the TAG amber stop codon (aselector codon) encountered in a transcript. The orthogonality of thistRNA/synthetase pair ensures that neither the tRNA nor the synthetasecross reacts with endogenous E. coli tRNAs or synthetases and that theunnatural amino acid gets incorporated only in response to TAG.

A search for orthogonal synthetases that have the ability tospecifically charge an orthogonal tRNA with 7-amino-coumarin alanine or7-hydroxy-coumarin alanine was undertaken. This search used protocolsthat have been previously described. A library of M. jannaschii tyrosyltRNA-synthetase mutants was generated by mutagenesis of the wild-type M.jannaschii tyrosyl tRNA-synthetase, where the mutagenesis consisted ofrandomizing six predicted active site residues based on the crystalstructure of other aminoacyl tRNA-synthetase molecules. The library hasa diversity of approximately 10⁹ species.

Following mutagenesis, the mutant synthetase library was passed throughmultiple rounds of positive and negative selection. This selectionyielded one synthetase clone that had the ability to charge the O-tRNAwith 7-amino-coumarin alanine or 7-hydroxy-coumarin alanine. Thatsynthetase clone was sequenced, and the amino acid sequence wasdetermined (see, Table 5, SEQ ID NO: 63). This synthetase mutant showsthe follow substitutions relative to the wild-type M. janaschiityrosyl-tRNA synthetase sequence: Y32R, L65A, H70M, D158N and L162T.

Additional data has been obtained demonstrating the selectiveincorporation of the coumarin alanine amino acids into proteins inresponse to a selector codon in an orthogonal translation systemcomprising the isolated synthetase species. This data includes, (a)expression studies where a myoglobin gene having a TAG selector codon atposition 4 is expressed only in the presence of the unnatural aminoacid; (b) the mutant myoglobin synthesized in the presence of theunnatural amino acid appears as a fluorescent band in an SDS-PAGE gelanalysis; and (c) the isolated mutant synthetase has been crystallized,and the co-crystal structure of the mutant synthetase in the presence ofthe unnatural amino acid is fluorescent.

Example 17 O-RS and O-tRNA Species for the Incorporation of UnnaturalAmino Acids

A variety of O-tRNA species can be used with the present invention, andthe invention is not limited to the use of any particular O-tRNA. Forexample, O-tRNA species that comprise the nucleotide sequence of SEQ IDNO: 1 or SEQ ID NO: 2 find use with the invention. With the teachingprovided herein, additional O-tRNA species can be constructed for usewith the invention.

Similarly, O-RS species are also provided (see, Table 5) for use inprotocols for the incorporation of unnatural amino acids, e.g., anunnatural amino acid selected from p-ethylthiocarbonyl-L-phenylalanine,p-(3-oxobutanoyl)-L-phenylalanine, 1,5-dansyl-alanine, 7-amino-coumarinalanine, 7-hydroxy-coumarin alanine, o-nitrobenzyl-serine,O-(2-nitrobenzyl)-L-tyrosine, p-carboxymethyl-L-phenylalanine,p-cyano-L-phenylalanine, m-cyano-L-phenylalanine, biphenylalanine,3-amino-L-tyrosine, bipyridylalanine,p-(2-amino-1-hydroxyethyl)-L-phenylalanine;p-isopropylthiocarbonyl-L-phenylalanine; 3-nitro-L-tyrosine andp-nitro-L-phenylalanine. The O-RS polypeptides of the invention includethose polypeptides that comprise the amino acid sequences provided inTable 5, SEQ ID NOS: 7-10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32,34, 36, 38, 40, 42, 44, 46, 50, 52-55, 57 and 59-63.

Examples of polynucleotides that encode O-RSs or portions thereof arealso provided. For example, polynucleotides that encode O-RS moleculesof the invention include SEQ ID NOS: 11, 13, 15, 17, 19, 21, 23, 25, 27,29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 51, 56, 58. However, it is notintended that the polynucleotides of the invention be limited to thoseprovided in Table 5. Indeed, any polynucleotide that encodes an O-RSamino acid sequence of the invention, e.g., SEQ ID NOS: 7-10, 12, 14,16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 50,52-55, 57 and 59-63, is also a feature of the invention.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims.

While the foregoing invention has been described in some detail forpurposes of clarity and understanding, it will be clear to one skilledin the art from a reading of this disclosure that various changes inform and detail can be made without departing from the true scope of theinvention. For example, all the techniques and apparatus described abovecan be used in various combinations. All publications, patents, patentapplications, and/or other documents cited in this application areincorporated by reference in their entirety for all purposes to the sameextent as if each individual publication, patent, patent application,and/or other document were individually indicated to be incorporated byreference for all purposes.

Example 18 Nucleotide and Amino Acid Sequences

This Example provides nucleotide and amino acid sequences for variouspolynucleotides and polypeptides, respectively. The sequences providedin Table 5 below are meant to provide examples only, and it is notintended that the invention be limited in any way by the sequencesprovided Table 5.

TABLE 5 Nucleotide and Amino Acid Sequences SEQ ID NO: DescriptionSEQUENCE  1 Methanococcus jannaschii-CCGGCGGUAGUUCAGCAGGGCAGAACGGCGGACUCUAAAUCCGsuppressor tyrosyl-tRNA_(CUA) CAUGGCGCUGGUUCAAAUCCGGCCCGCCGGACCAmutRNA^(Tyr) _(CUA)  2 E. coliGCCCGGAUGGUGGAAUCGGUAGACACAAGGGAUUCUAAAUCCC suppressor tRNA^(Leu5)_(CUA) UCGGCGUUCGCGCUGUGCGGGUUCAAGUCCCGCUCCGGGUACC A  3Wild-type Methanococcus MDEFEMIKRNTSEIISEEELREVLKKDEKSAYIGFEPSGKIHLjannaschii tyrosyl-tRNA GHYLQIKKEIDLQNAGFDIIILLADLHAYLNQKGELDEIRKIGsynthetase (MjTyrRS) DYNKKVFEAMGLKAKYVYGSEFQLDKDYTLNVYRLALKTTLKRamino acid sequence ARRSMELIAREDENPKVAEVIYPIMQVNDIHYLGVDVAVGGMEQRKIHMLARELLPKKVVCIHNPVLTGLDGEGKMSSSKGNFIAVDDSPEEIRAKIKKAYCPAGVVEGNPIMEIAKYFLEYPLTIKRPEKFGGDLTVNSYEELESLFKNKELHPMDLKNAVAEELIKILEP IRKRL  4Wild-type Methanococcus ATGGACGAATTTGAAATGATAAAGAGAAACACATCTGAAATTATCAGCjannaschii tyrosyl-tRNA GAGGAAGAGTTAAGAGAGGTTTTAAAAAAAGATGAAAAATCTGCTTACsynthetase (MjTyrRS) ATAGGTTTTGAACCAAGTGGTAAAATACATTTAGGGCATTATCTCCAAnucleotide sequence ATAAAAAAGATGATTGATTTACAAAATGCTGGATTTGATATAATTATATTGTTGGCTGATTTACACGCCTATTTAAACCAGAAAGGAGAGTTGGATGAGATTAGAAAAATAGGAGATTATAACAAAAAAGTTTTTGAAGCAATGGGGTTAAAGGCAAAATATGTTTATGGAAGTGAATTCCAGCTTGATAAGGATTATACACTGAATGTCTATAGATTGGCTTTAAAAACTACCTTAAAAAGAGCAAGAAGGAGTATGGAACTTATAGCAAGAGAGGATGAAAATCCAAAGGTTGCTGAAGTTATcTATccAATAATGcAGGTTAATGATATTcATTATTTAGGCGTTGATGTTGCAGTTGGAGGGATGGAGCAGAGAAAAATACACATGTTAGCAAGGGAGCTTTTACCAAAAAAGGTTGTTTGTATTCACAACCCTGTCTTAACGGGTTTGGATGGAGAAGGAAAGATGAGTTCTTCAAAAGGGAATTTTATAGCTGTTGATGACTCTCCAGAAGAGATTAGGGCTAAGATAAAGAAAGCATACTGCCCAGCTGGAGTTGTTGAAGGAAATCCAATAATGGAGATAGCTAAATACTTCCTTGAATATCCTTTAACCATAAAAAGGCCAGAAAAATTTGGTGGAGATTTGACAGTTAATAGCTATGAGGAGTTAGAGAGTTTATTTAAAAATAAGGAATTGCATCCAATGGATTTAAAAAATGCTGTAGCTGAAGAACTTATAAAGATTTTAGAGCCAATTAGAAAG AGATTA  5Wild-type E. coli MQEQYRPEEIESKVQLHWDEKRTFEVTEDESKEKYYCLSMLPYleucyl-tRNA PSGRLHMGHVRNYTIGDVIARYQRMLGKNVLQPIGWDAFGLPAsynthetase (EcLeuRS) EGAAVKNNTAPAPWTYDNIAYMKNQLKMLGFGYDWSRELATCTamino acid sequence PEYYRWEQKFFTELYKKGLVYKKTSAVNWCPNDQTVLANEQVIDGCCWRCDTKVERKEIPQWFIKITAYADELLNDLDKLDHWPDTVKTMQRNWIGRSEGVEITFNVNDYDNTLTVYTTRPDTFMGCTYLAVAAGHPLAQKAAENNPELAAFIDECRNTKVAEAEMATMEKKGVDTGFKAVHPLTGEEIPVWAANFVLMEYGTGAVMAVPGHDQRDYEFASKYGLNIKPVILAADGSEPDLSQQALTEKGVLFNSGEFNGLDHEAAFNAIADKLTAMGVGERKVNYRLRDWGVSRQRYWGAPIPMVTLEDGTVMPTPDDQLPVILPEDVVMDGITSPIKADPEWAKTTVNGMPALRETDTFDTFMESSWYYARYTCPQYKEGMLDSEAANYWLPVDIYIGGIEHAIMHLLYFRFFHKLMRDAGMVNSDEPAKQLLCQGMVLADAFYYVGENGERNWVSPVDAIVERDEKGRIVKAKDAAGHELVYTGMSKMSKSKNNGIDPQVMVERYGADTVRLFMMFASPADMTLEWQESGVEGANRFLKRVWKLVYEHTAKGDVAALNVDALTENQKALRRDVHKTIAKVTDDIGRRQTFNTAIAAIMELMNKLAKAPTDCEQDRALMQEALLAVVRMLNPFTPHICFTLWQELKGEGDIDNAPWPVADEKAMVEDSTLVVVQVNGKVRAKITVPVDATEEQVRERAGQEHLVAKYLDGVTVRKVIYVPGKLLNLVVG  6 Wild-type E. coliATGCAAGAGCAATACCGCCCGGAAGAGATAGAATCCAAAGTAC leucyl-tRNAAGCTTCATTGGGATGAGAAGCGCACATTTGAAGTAACCGAAGA synthetase (EcLeuRS)CGAGAGCAAAGAGAAGTATTACTGCCTGTCTATGCTTCCCTAT nucleotide sequenceCCTTCTGGTCGACTACACATGGGCCACGTACGTAACTACACCATCGGTGACGTGATCGCCCGCTACCAGCATATGCTGGGCAAAAACGTCCTGCACCCGATCGGCTGGGACGCGTTTGGTCTGCCTGCGGAAGGCGCGGCGGTGAAAAACAACACCGCTCCGGCACCGTGGACGTACGACAACATCGCGTATATGAAAAACCAGCTCAAAATGCTGGGCTTTGGTTATGACTGGAGCCGCGAGCTGGCAACCTGTACGCCGGAATACTACCGTTGGGAACAGAAATTCTTCACCGAGCTGTATAAAAAAGGCCTGGTATATAAGAAGACTTCTGCGGTCAACTGGTGCCCGAACGACCAGACCGTACTGGCGAACGAACAAGTTATCGACGGCTGCTGCTGGCGCTGCGATACCAAAGTTGAACGTAAAGAGATCCCGCAGTGGTTTATCAAAATCAATGCTTACGCTGACGAGCTGCTCAACGATCTGGATAAACTGGATCACTGGCCAGACACCGTTAAAACCATGCAGCGTAACTGGATCGGTCGTTCCGAAGGCGTGGAGATCACCTTCAACGTTAACGACTATGACAACACGCTGACCGTTTACACTACCCGCCCGGACACCTTTATGGGTTGTACCTACCTGGCGGTACGTGCGGGTCATCCGCTGGCGCAGAAAGCGGCGGAAAATAATCCTGAACTGGCGGCCTTTATTGACGAATGCCGTAACACCAAAGTTGCCGAAGCTGAAATGGCGACGATGGAGAAAAAAGGCGTCGATACTGGCTTTAAAGCGGTTCACCCATTAACGGGCGAAGAAATTCCCGTTTGGGCAGCAAACTTCGTATTGATGGAGTACGGCACGGGCGCAGTTATGGCGGTACCGGGGCACGACCAGCGCGACTACGAGTTTGCCTCTAAATACGGCCTGAACATCAAACCGGTTATCCTGGCAGCTGACGGCTCTGAGCCAGATCTTTCTCAGCAAGCCCTGACTGAAAAAGGCGTGCTGTTCAACTCTGGCGAGTTCAACGGTCTTGACCATGAAGCGGCCTTCAACGCCATCGCCGATAAACTGACTGCGATGGGCGTTGGCGAGCGTAAAGTGAACTACCGCCTGCGCGACTGGGGTGTTTCCCGTCAGCGTTACTGGGGCGCGCCGATTCCGATGGTGACGCTGGAAGACGGTACCGTAATGCCGACCCCGGACGACCAGCTGCCGGTGATCCTGCCGGAAGATGTGGTAATGGACGGCATTACCAGCCCGATTAAAGCAGATCCGGAGTGGGCGAAAACTACCGTTAACGGTATGCCAGCACTGCGTGAAACCGACACTTTCGACACCTTTATGGAGTCCTCCTGGTACTATGCGCGCTACACTTGCCCGCAGTACAAAGAAGGTATGCTGGATTCCGAAGCGGCTAACTACTGGCTGCCGGTGGATATCTACATTGGTGGTATTGAACACGCCATTATGCACCTGCTCTACTTCCGCTTCTTCCACAAACTGATGCGTGATGCAGGCATGGTGAACTCTGACGAACCAGCGAAACAGTTGCTGTGTCAGGGTATGGTGCTGGCAGATGCCTTCTACTATGTTGGCGAAAACGGCGAACGTAACTGGGTTTCCCCGGTTGATGCTATCGTTGAACGTGACGAGAAAGGCCGTATCGTGAAAGCGAAAGATGCGGCAGGCCATGAACTGGTTTATACCGGCATGAGCAAAATGTCCAAGTCGAAGAACAACGGTATCGACCCGCAGGTGATGGTTGAACGTTACGGCGCGGACACCGTTCGTCTGTTTATGATGTTTGCTTCTCCGGCTGATATGACTCTCGAATGGCAGGAATCCGGTGTGGAAGGGGCTAACCGCTTCCTGAAACGTGTCTGGAAACTGGTTTACGAGCACACAGCAAAAGGTGATGTTGCGGCACTGAACGTTGATGCGCTGACTGAAAATCAGAAAGCGCTGCGTCGCGATGTGCATAAAACGATCGCTAAAGTGACCGATGATATCGGCCGTCGTCAGACCTTCAACACCGCAATTGCGGCGATTATGGAGCTGATGAACAAACTGGCGAAAGCACCAACCGATGGCGAGCAGGACCGCGCTCTGATGCAGGAAGCACTGCTGGCCGTTGTCCGTATGCTTAACCCGTTCACCCCGCACATCTGCTTCACGCTGTGGCAGGAACTGAAAGGCGAAGGCGATATCGACAACGCGCCGTGGCCGGTTGCTGACGAAAAAGCGATGGTGGAAGACTCCACGCTGGTCGTGGTGCAGGTTAACGGTAAAGTCCGTGCCAAAATCACCGTTCCGGTGGACGCAACGGAAGAACAGGTTCGCGAACGTGCTGGCCAGGAACATCTGGTAGCAAAATATCTTGATGGCGTTACTGTACGTAAAGTGATTTACGTACCAGGTAAACTCCTCAATCTGGTCGTTGGC TAA  7 3-nitro-L-tyrosineMDEFEMIKRNTSEIISEEELREVLKKDEKSAYIGFEPSGKIHL aminoacyl-GHYLQIKKMIDLQNAGFDIIILLVDLVAYLNQKGELDEIRKI tRNA synthetaseGDYNKKVFEAMGLKAKYVYGSEFQLDKDYTLNVYRLALKTTLK isolates-A, B, C,RARRSMELIAREDENPKVAEVIYPIMQVNDIHYLGVDVAVGGM E and G amino acidEQRKIHMLARELLPKKVVCIHNPVLTGLDGEGKMSSSKGNFIA sequence (derivedVDDSPEEIRAKIKKAYCPAGVVEGNPIMEIAKYFLEYPLTIKR from wild-typePEKFGGDLTVNSYEELESLFKNKELHPMDLKNAVAEELIKILE Methanococcus PIRKRLjannaschii tyrosyl tRNA-synthetase), each having amino acid changes:A67V, H70V  8 3-nitro-L-tyrosineMDEFEMIKRNTSEIISEEELREVLKKDEKSASIGFEPSGKIHL aminoacyl-GHYLQIKKMIDLQNAGFDIIILLTDLNAYLNQKGELDEIRKI tRNA synthetaseGDYNKKVFEANGLKAKYVYGSEFQLDKDYTLNVYRLALKTTLK isolate-D aminoRARRSMELIAREDENPKVAEVIYPIMQVNDIHYLGVDVTVGGM acid sequenceEQRKIHMLARELLPKKVVCIHNPVLTGLDGEGKMSSSKGNFIA (derived fromVDDSPEEIRAKIKKAYCPAGVVEGNPIMEIAKYFLEYPLTIKR wild-typePEKFGGDLTVNSYEELESLFKNKELHPMDLKNAVAEELIKILE Methanococcus PIRKRLjannaschii tyrosyl tRNA synthetase), having amino acid changes: Y32S,A67T, H70N, A167T  9 3-nitro-L-tyrosineMDEFEMIKRNTSEIISEEELREVLKKDEKSAYIGFEPSGKIHL aminoacyl-GHYLQIKKMIDLQNAGFDIIILLPDLHAYLNQKGELDEIRKIG tRNA synthetaseDYNKKVFEAMGLKAKYVYGSEFQLDKDYTLNVYRLALKTTLKR isolate-F aminoARRSMELIAREDENPKVAEVIYPIMQVNDIHYLGVDVGVGGME acid sequenceQRKIHMLARELLPKKVVCIHNPVLTGLDGEGKMSSSKGNFIAV (derived fromDDSPEEIRAKIKKAYCPAGVVEGNPIMEIAKYFLEYPLTIKRP wild-typeEKFGGDLTVNSYEELESLFKNKELHPMDLKNAVAEELIKILEP Methanococcus IRKRLjannaschii tyrosyl tRNA-synthetase), having amino acid changes: Y32A,A67P, A167G 10 p-nitro-L-phenylalanineMDEFEMIKRNTSEIISEEELREVLKKDEKSALIGFEPSGKIHL aminoacyl-tRNA synthetaseGHYLQIKKMIDLQNAGFDIIILLADLHAYLNQKGELDEIRKIG amino acid sequenceDYNKKVFEAMGLKAKYVYGSSFQLDKDYTLNVYRLALKTTLKR (derived from wild-typeARRSMELIAREDENPKVAEVIYPIMQVNPLNYEGVDVAVGGM MethanococcusEQRKIHMLARELLPKKVVCIHNPVLTGLDGEGKMSSSKGNFIA jannaschii tyrosylVDDSPEEIRAKIKKAYCPAGVVEGNPIMEIAKYFLEYPLTIKR tRNA-synthetase),PEKFGGDLTVNSYEELESLFKNKELHPMDLKNAVAEELIKILE having amino acid PIRKRLchanges: Tyr32Leu, Glu107Ser, Asp158Pro, Ile159Leu, His160Asn, Leu162Glu11 p-nitro-L-phenylalanine ATGGACGAATTTGAAATGATAAAGAGAAACACATCTGAAATTAaminoacyl-tRNA synthetase TCAGCGAGGAAGAGTTAAGAGAGGTTTTAAAAAAAGATGAAAAnucleotide sequence ATCTGCTCTGATAGGTTTTGAACCAAGTGGTAAAATACATTTAGGGCATTATCTCCAAATAAAAAAGATGATTGATTTACAAAATGCTGGATTTGATATAATTATATTGTTGGCTGATTTACACGCCTATTTAAACCAGAAAGGAGAGTTGGATGAGATTAGAAAAATAGGAGATTATAACAAAAAAGTTTTTGAAGCAATGGGGTTAAAGGCAAAATATGTTTATGGAAGTTCGTTCCAGCTTGATAAGGATTATACACTGAATGTCTATAGATTGGCTTTAAAAACTACCTTAAAAAGAGCAAGAAGGAGTATGGAACTTATAGCAAGAGAGGATGAAAATCCAAAGGTTGCTGAAGTTATCTATCCAATAATGCAGGTTAATCCTCTTAATTATGAGGGCGTTGATGTTGCAGTTGGAGGGATGGAGCAGAGAAAAATACACATGTTAGCAAGGGAGCTTTTACCAAAAAAGGTTGTTTGTATTCACAACCCTGTCTTAACGGGTTTGGATGGAGAAGGAAAGATGAGTTCTTCAAAAGGGAATTTTATAGCTGTTGATGACTCTCCAGAAGAGATTAGGGCTAAGATAAAGAAAGCATACTGCCCAGCTGGAGTTGTTGAAGGAAATCCAATAATGGAGATAGCTAAATACTTCCTTGAATATCCTTTAACCATAAAAAGGCCAGAAAAATTTGGTGGAGATTTGACAGTTAATAGCTATGAGGAGTTAGAGAGTTTATTTAAAAATAAGGAATTGCATCCAATGGATTTAAAAAATGCTGTAGCTGAAGAACTTATAAAGATTTTAGAGCCA ATTAGAAAGAGATTATAA 123-amino-L-tyrosine MDEFEMIKRNTSEIISEEELREVLKKDEKSAQIGFEPSGKIHLaminoacyl-tRNA GHYLQIKKMIDLQNAGFDIIIELADLHAYLNQKGELDEIRKIGsynthetase amino acid DYNKKVFEAMGLKAKYVYGSEGLLDKDYTLNVYRLALKTTLKRsequence (derived from ARRSMELIAREDENPKVAEVIYPIMQVNSIHYTGVDVAVGGMEwild-type Methanococcus QRKIHMLARELLPKKVVCIHNPVLTGLDGEGKMSSSKGNFIAVjannaschii tyrosyl tRNA- DDSPEEIRAKIKKAYCPAGVVEGNPIMEIAKYFLEYPLTIKRPsynthetase) EKFGGDLTVNSYEELESLFKNKELHPMDLKNAVAEELIKILEP IRKRL 133-amino-L-tyrosine ATGGACGAATTTGAAATGATAAAGAGAAACACATCTGAAATTAaminoacyl-tRNA TCAGCGAGGAAGAGTTAAGAGAGGTTTTAAAAAAAGATGAAAAsynthetase nucleotide ATCTGCTCAGATAGGTTTTGAACCAAGTGGTAAAATACATTTAsequence GGGCATTATCTCCAAATAAAAAAGATGATTGATTTACAAAATGCTGGATTTGATATAATTATAGAGTTGGCTGATTTACACGCCTATTTAAACCAGAAAGGAGAGTTGGATGAGATTAGAAAAATAGGAGATTATAACAAAAAAGTTTTTGAAGCAATGGGGTTAAAGGCAAAATATGTTTATGGAAGTGAAGGTTTGCTTGATAAGGATTATACACTGAATGTCTATAGATTGGCTTTAAAAACTACCTTAAAAAGAGCAAGAAGGAGTATGGAACTTATAGCAAGAGAGGATGAAAATCCAAAGGTTGCTGAAGTTATCTATCCAATAATGCAGGTTAATTCTATTCATTATACTGGCGTTGATGTTGCAGTTGGAGGGATGGAGCAGAGAAAAATACACATGTTAGCAAGGGAGCTTTTACCAAAAAAGGTTGTTTGTATTCACAACCCTGTCTTAACGGGTTTGGATGGAGAAGGAAAGATGAGTTCTTCAAAAGGGAATTTTATAGCTGTTGATGACTCTCCAGAAGAGATTAGGGCTAAGATAAAGAAAGCATACTGCCCAGCTGGAGTTGTTGAAGGAAATCCAATAATGGAGATAGCTAAATACTTCCTTGAATATCCTTTAACCATAAAAAGGCCAGAAAAATTTGGTGGAGATTTGACAGTTAATAGCTATGAGGAGTTAGAGAGTTTATTTAAAAATAAGGAATTGCATCCAATGGATTTAAAAAATGCTGTAGCTGAAGAACTTATAAAGATTTTAGAGCCA ATTAGAAAGAGATTATAA 14p-carboxymethyl-L- MDEFEMIKRNTSEIISEEELREVLKKDEKSAAIGFEPSGKIHLphenylalanine GHYLQIKKMIDLQNAGFDIIISLADLHAYLNQKGELDEIRKIG aminoacyl-tRNADYNKKVFEAMGLKAKYVYGSERNLDKDYTLNVYRLALKTTLKR synthetase cloneARRSMELIAREDENPKVAEVIYPIMQVNSIHYHGVDVAVGGME #1 amino acidQRKIHMLARELLPKKVVCIHNPVLTGLDGEGKMSSSKGNFIAV sequence (derivedDDSPEEIRAKIKKAYCPAGVVEGNPIMEIAKYFLEYPLTIKRP from wild-typeEKFGGDLTVNSYEELESLFKNKELHPMDLKNAVAEELIKILEP Methanococcus IRKRLjannaschii tyrosyl tRNA-synthetase) 15 p-carboxymethyl-L-ATGGACGAATTTGAAATGATAAAGAGAAACACATCTGAAATTA phenylalanineTCAGCGAGGAAGAGTTAAGAGAGGTTTTAAAAAAAGATGAAAA aminoacyl-tRNAATCTGCTGCGATAGGTTTTGAACCAAGTGGTAAAATACATTTA synthetase cloneGGGCATTATCTCCAAATAAAAAAGATGATTGATTTACAAAATG #1 nucleotideCTGGATTTGATATAATTATATCGTTGGCTGATTTACACGCCTA sequenceTTTAAACCAGAAAGGAGAGTTGGATGAGATTAGAAAAATAGGAGATTATAACAAAAAAGTTTTTGAAGCAATGGGGTTAAAGGCAAAATATGTTTATGGAAGTGAACGTAATCTTGATAAGGATTATACACTGAATGTCTATAGATTGGCTTTAAAAACTACCTTAAAAAGAGCAAGAAGGAGTATGGAACTTATAGCAAGAGAGGATGAAAATCCAAAGGTTGCTGAAGTTATCTATCCAATAATGCAGGTTAATTCTATTCATTATCATGGCGTTGATGTTGCAGTTGGAGGGATGGAGCAGAGAAAAATACACATGTTAGCAAGGGAGCTTTTACCAAAAAAGGTTGTTTGTATTCACAACCCTGTCTTAACGGGTTTGGATGGAGAAGGAAAGATGAGTTCTTCAAAAGGGAATTTTATAGCTGTTGATGACTCTCCAGAAGAGATTAGGGCTAAGATAAAGAAAGCATACTGCCCAGCTGGAGTTGTTGAAGGAAATCCAATAATGGAGATAGCTAAATACTTCCTTGAATATCCTTTAACCATAAAAAGGCCAGAAAAATTTGGTGGAGATTTGACAGTTAATAGCTATGAGGAGTTAGAGAGTTTATTTAAAAATAAGGAATTGCATCCAATGGATTTAAAAAATGCTGTAGCTGAAGAACTTATAAAGATTTTAGAGCCA ATTAGAAAGAGATTATAA 16p-carboxymethyl-L- MDEFEMIKRNTSEIISEEELREVLKKDEKSASIGFEPSGKIHLphenylalanine GHYLQIKKMIDLQNAGFDIIIALADLHAYLNQKGELDEIRKIG aminoacyl-tRNADYNKKVFEAMGLKAKYVYGSENYLDKDYTLNVYRLALKTTLKR synthetase cloneARRSMELIAREDENPKVAEVIYPIMQVNGIHYKGVDVAVGGME #2 amino acidQRKIHMLARELLPKKVVCIHNPVLTGLDGEGKMSSSKGNFIAV sequence (derivedDDSPEEIRAKIKKAYCPAGVVEGNPIMEIAKYFLEYPLTIKRP from wild-typeEKFGGDLTVNSYEELESLFKNKELHPMDLKNAVAEELIKILEP Methanococcus IRKRLjannaschii tyrosyl tRNA-synthetase) 17 p-carboxymethyl-L-ATGGACGAATTTGAAATGATAAAGAGAAACACATCTGAAATTA phenylalanineTCAGCGAGGAAGAGTTAAGAGAGGTTTTAAAAAAAGATGAAAA aminoacyl-tRNAATCTGCTTCTATAGGTTTTGAACCAAGTGGTAAAATACATTTA synthetase cloneGGGCATTATCTCCAAATAAAAAAGATGATTGATTTACAAAATG #2 nucleotideCTGGATTTGATATAATTATAGCTTTGGCTGATTTACACGCCTA sequenceTTTAAACCAGAAAGGAGAGTTGGATGAGATTAGAAAAATAGGAGATTATAACAAAAAAGTTTTTGAAGCAATGGGGTTAAAGGCAAAATATGTTTATGGAAGTGAAAATTATCTTGATAAGGATTATACACTGAATGTCTATAGATTGGCTTTAAAAACTACCTTAAAAAGAGCAAGAAGGAGTATGGAACTTATAGCAAGAGAGGATGAAAATCCAAAGGTTGCTGAAGTTATCTATCCAATAATGCAGGTTAATGGTATTCATTATAAGGGCGTTGATGTTGCAGTTGGAGGGATGGAGCAGAGAAAAATACACATGTTAGCAAGGGAGCTTTTACCAAAAAAGGTTGTTTGTATTCACAACCCTGTCTTAACGGGTTTGGATGGAGAAGGAAAGATGAGTTCTTCAAAAGGGAATTTTATAGCTGTTGATGACTCTCCAGAAGAGATTAGGGCTAAGATAAAGAAAGCATACTGCCCAGCTGGAGTTGTTGAAGGAAATCCAATAATGGAGATAGCTAAATACTTCCTTGAATATCCTTTAACCATAAAAAGGCCAGAAAAATTTGGTGGAGATTTGACAGTTAATAGCTATGAGGAGTTAGAGAGTTTATTTAAAAATAAGGAATTGCATCCAATGGATTTAAAAAATGCTGTAGCTGAAGAACTTATAAAGATTTTAGAGCCA ATTAGAAAGAGATTATAA 18p-carboxymethyl-L- MDEFEMIKRNTSEIISEEELREVLKKDEKSASIGFEPSGKIHLphenylalanine GHYLQIKKMIDLQNAGFDIIIALADLHAYLNQKGELDEIRKIG aminoacyl-tRNADYNKKVFEAMGLKAKYVYGSERQLDKDYTLNVYRLALKTTLKR synthetase cloneARRSMELIAREDENPKVAEVIYPIMQVNGIHYKGVDVAVGGME #3 amino acidQRKIHMLARELLPKKVVCIHNPVLTGLDGEGKMSSSKGNFIAV sequence (derivedDDSPEEIRAKIKKAYCPAGVVEGNPIMEIAKYFLEYPLTIKRP from wild-typeEKFGGDLTVNSYEELESLFKNKELHPMDLKNAVAEELIKILEP Methanococcus IRKRLjannaschii tyrosyl tRNA-synthetase) 19 p-carboxymethyl-L-ATGGACGAATTTGAAATGATAAAGAGAAACACATCTGAAATTA phenylalanineTCAGCGAGGAAGAGTTAAGAGAGGTTTTAAAAAAAGATGAAAA aminoacyl-tRNAATCTGCTTCGATAGGTTTTGAACCAAGTGGTAAAATACATTTA synthetase cloneGGGCATTATCTCCAAATAAAAAAGATGATTGATTTACAAAATG #3 nucleotideCTGGATTTGATATAATTATAGCGTTGGCTGATTTACACGCCTA sequenceTTTAAACCAGAAAGGAGAGTTGGATGAGATTAGAAAAATAGGAGATTATAACAAAAAAGTTTTTGAAGCAATGGGGTTAAAGGCAAAATATGTTTATGGAAGTGAACGTCAGCTTGATAAGGATTATACACTGAATGTCTATAGATTGGCTTTAAAAACTACCTTAAAAAGAGCAAGAAGGAGTATGGAACTTATAGCAAGAGAGGATGAAAATCCAAAGGTTGCTGAAGTTATCTATCCAATAATGCAGGTTAATGGTATTCATTATAAGGGCGTTGATGTTGCAGTTGGAGGGATGGAGCAGAGAAAAATACACATGTTAGCAAGGGAGCTTTTACCAAAAAAGGTTGTTTGTATTCACAACCCTGTCTTAACGGGTTTGGATGGAGAAGGAAAGATGAGTTCTTCAAAAGGGAATTTTATAGCTGTTGATGACTCTCCAGAAGAGATTAGGGCTAAGATAAAGAAAGCATACTGCCCAGCTGGAGTTGTTGAAGGAAATCCAATAATGGAGATAGCTAAATACTTCCTTGAATATCCTTTAACCATAAAAAGGCCAGAAAAATTTGGTGGAGATTTGACAGTTAATAGCTATGAGGAGTTAGAGAGTTTATTTAAAAATAAGGAATTGCATCCAATGGATTTAAAAAATGCTGTAGCTGAAGAACTTATAAAGATTTTAGAGCCA ATTAGAAAGAGATTATAA 20p-carboxymethyl-L- MDEFEMIKRNTSEIISEEELREVLKKDEKSASIGFEPSGKIHLphenylalanine GHYLQIKKMIDLQNAGFDIIIALADLHAYLNQKGELDEIRKIG aminoacyl-tRNADYNKKVFEAMGLKAKYVYGSEAQLDKDYTLNVYRLALKTTLKR synthetase cloneARRSMELIAREDENPKVAEVIYPIMQVNGIHYKGVDVAVGGME #4 amino acidQRKIHMLARELLPKKVVCIHNPVLTGLDGEGKMSSSKGNFIAV sequence (derivedDDSPEEIRAKIKKAYCPAGVVEGNPIMEIAKYFLEYPLTIKRP from wild-typeEKFGGDLTVNSYEELESLFKNKELHPMDLKNAVAEELIKILEP Methanococcus IRKRLjannaschii tyrosyl tRNA-synthetase) 21 p-carboxymethyl-L-ATGGACGAATTTGAAATGATAAAGAGAAACACATCTGAAATTA phenylalanineTCAGCGAGGAAGAGTTAAGAGAGGTTTTAAAAAAAGATGAAAA aminoacyl-tRNAATCTGCTTCGATAGGTTTTGAACCAAGTGGTAAAATACATTTA synthetase cloneGGGCATTATCTCCAAATAAAAAAGATGATTGATTTACAAAATG #4 nucleotideCTGGATTTGATATAATTATAGCGTTGGCTGATTTACACGCCTA sequenceTTTAAACCAGAAAGGAGAGTTGGATGAGATTAGAAAAATAGGAGATTATAACAAAAAAGTTTTTGAAGCAATGGGGTTAAAGGCAAAATATGTTTATGGAAGTGAAGCGCAGCTTGATAAGGATTATACACTGAATGTCTATAGATTGGCTTTAAAAACTACCTTAAAAAGAGCAAGAAGGAGTATGGAACTTATAGCAAGAGAGGATGAAAATCCAAAGGTTGCTGAAGTTATCTATCCAATAATGCAGGTTAATGGTATTCATTATAAGGGCGTTGATGTTGCAGTTGGAGGGATGGAGCAGAGAAAAATACACATGTTAGCAAGGGAGCTTTTACCAAAAAAGGTTGTTTGTATTCACAACCCTGTCTTAACGGGTTTGGATGGAGAAGGAAAGATGAGTTCTTCAAAAGGGAATTTTATAGCTGTTGATGACTCTCCAGAAGAGATTAGGGCTAAGATAAAGAAAGCATACTGCCCAGCTGGAGTTGTTGAAGGAAATCCAATAATGGAGATAGCTAAATACTTCCTTGAATATCCTTTAACCATAAAAAGGCCAGAAAAATTTGGTGGAGATTTGACAGTTAATAGCTATGAGGAGTTAGAGAGTTTATTTAAAAATAAGGAATTGCATCCAATGGATTTAAAAAATGCTGTAGCTGAAGAACTTATAAAGATTTTAGAGCCA ATTAGAAAGAGATTATAA 22p-carboxymethyl-L- MDEFEMIKRNTSETTSEEELREVLKKDEKSASIGFEPSGKIHLphenylalanine GHYLQIKKMIDLQNAGFDIIIALADLHAYLNQKGELDEIRKIG aminoacyl-tRNADYNKKVFEAMGLKAKYVYGSEKHLDKDYTLNVYRLALKTTLKR synthetase cloneARRSMELIAREDENPKVAEVIYPIMQVNGIHYKGVDVAVGGME #5 amino acidQRKIHMLARELLPKKVVCIHNPVLTGLDGEGKMSSSKGNFIAV sequence (derivedDDSPEETRAKIKKAYCPAGVVEGNPIMEIAKYFLEYPLTIKRP from wild-typeEKFGGDLTVNSYEELESLFKNKELHPMDLKNAVAEELIKILEP Methanococcus IRKRLjannaschii tyrosyl tRNA-synthetase) 23 p-carboxymethyl-L-ATGGACGAATTTGAAATGATAAAGAGAAACACATCTGAAATTA phenylalanineTCAGCGAGGAAGAGTTAAGAGAGGTTTTAAAAAAAGATGAAAA aminoacyl-tRNAATCTGCTTCTATAGGTTTTGAACCAAGTGGTAAAATACATTTA synthetase cloneGGGCATTATCTCCAAATAAAAAAGATGATTGATTTACAAAATG #5 nucleotideCTGGATTTGATATAATTATAGCGTTGGCTGATTTACACGCCTA sequenceTTTAAACCAGAAAGGAGAGTTGGATGAGATTAGAAAAATAGGAGATTATAACAAAAAAGTTTTTGAAGCAATGGGGTTAAAGGCAAAATATGTTTATGGAAGTGAAAAGCATCTTGATAAGGATTATACACTGAATGTCTATAGATTGGCTTTAAAAACTACCTTAAAAAGAGCAAGAAGGAGTATGGAACTTATAGCAAGAGAGGATGAAAATCCAAAGGTTGCTGAAGTTATCTATCCAATAATGCAGGTTAATGGCAGAGAAAAATACACATGTTAGCAAGGGAGCTTTTACCAAAAAAGGTTGTTTGTATTCACAACCCTGTCTTAACGGGTTTGGATGGAGAAGGAAAGATGAGTTCTTCAAAAGGGAATTTTATAGCTGTTGATGACTCTCCAGAAGAGATTAGGGCTAAGATAAAGAAAGCATACTGCCCAGCTGGAGTTGTTGAAGGAAATCCAATAATGGAGATAGCTAAATACTTCCTTGAATATCCTTTAACCATAAAAAGGCCAGAAAAATTTGGTGGAGATTTGACAGTTAATAGCTATGAGGAGTTAGAGAGTTTATTTAAAAATAAGGAATTGCATCCAATGGATTTAAAAAATGCTGTAGCTGAAGAACTTATAAAGATTTTAGAGCCA ATTAGAAAGAGATTATAA 24biphenylalanine MDEFEMIKRNTSEIISEEELREVLKKDEKSAAIGFEPSGKIHLaminoacyl-tRNA GHYLQIKKMIDLQNAGEDIIIGLADLHAYLNQKGELDEIRKIGsynthetase clone DYNKKVFEAMGLKAKYVYGSEEPLDKDYTLNVYRLALKTTLKR#1 amino acid ARRSMELIAREDENPKVAEVIYPIMQVNCIHYHGVDVAVGGMEsequence (derived QRKIHMLARELLPKKVVCIHNPVLTGLDGEGKMSSSKGNFIAVfrom wild-type DDSPEEIRAKIKKAYCPAGVVEGNPIMEIAKYFLEYPLTIKRP MethanococcusEKFGGDLTVNSYEELESLFKNKELHPMDLKNAVAEELIKILEP jannaschii tyrosyl IRKRLtRNA-synthetase) 25 biphenylalanineATGGACGAATTTGAAATGATAAAGAGAAACACATCTGAAATTA aminoacyl-tRNATCAGCGAGGAAGAGTTAAGAGAGGTTTTAAAAAAAGATGAAAA synthetase cloneATCTGCTGCTATAGGTTTTGAACCAAGTGGTAAAATACATTTA #1 nucleotideGGGCATTATCTCCAAATAAAAAAGATGATTGATTTACAAAATG sequenceCTGGATTTGATATAATTATAGGGTTGGCTGATTTACACGCCTATTTAAACCAGAAAGGAGAGTTGGATGAGATTAGAAAAATAGGAGATTATAACAAAAAAGTTTTTGAAGCAATGGGGTTAAAGGCAAAATATGTTTATGGAAGTGAAGAGCCGCTTGATAAGGATTATACACTGAATGTCTATAGATTGGCTTTAAAAACTACCTTAAAAAGAGCAAGAAGGAGTATGGAACTTATAGCAAGAGAGGATGAAAATCCAAAGGTTGCTGAAGTTATCTATCCAATAATGCAGGTTAATTGTATTCATTATCATGGCGTTGATGTTGCAGTTGGAGGGATGGAGCAGAGAAAAATACACATGTTAGCAAGGGAGCTTTTACCAAAAAAGGTTGTTTGTATTCACAACCCTGTCTTAACGGGTTTGGATGGAGAAGGAAAGATGAGTTCTTCAAAAGGGAATTTTATAGCTGTTGATGACTCTCCAGAAGAGATTAGGGCTAAGATAAAGAAAGCATACTGCCCAGCTGGAGTTGTTGAAGGAAATCCAATAATGGAGATAGCTAAATACTTCCTTGAATATCCTTTAACCATAAAAAGGCCAGAAAAATTTGGTGGAGATTTGACAGTTAATAGCTATGAGGAGTTAGAGAGTTTATTTAAAAATAAGGAATTGCATCCAATGGATTTAAAAAATGCTGTAGCTGAAGAACTTATAAAGATTTTAGAGCCA ATTAGAAAGAGATTATAA 26biphenylalanine MDEFEMIKRNTSEIISEEELREVLKKDEKSALIGFEPSGKIHLaminoacyl-tRNA GHYLQIKKMIDLQNAGEDIIITLADLSAYLNQKGELDEIRKIGsynthetase clone DYNKKVFEAMGLKAKYVYGSEFQLDKDYTLNVYRLALKTTLKR#2 amino acid ARRSMELIAREDENPKVAEVIYPIMGVNVIHYHGVDVAVGGMEsequence (derived QRKIHMLARELLPKKVVCIHNPVLTGLDGEGKMSSSKGNFIAVfrom wild-type DDSPEEIRAKIKKAYCPAGVVEGNPIMEIAKYFLEYPLTIKRP MethanococcusEKEGGDLTVNSYEELESLEKNKELHPMDLKNAVAEELIKILEP jannaschii tyrosyl IRKRLtRNA-synthetase) 27 biphenylalanineATGGACGAATTTGAAATGATAAAGAGAAACACATCTGAAATTA aminoacyl-tRNATCAGCGAGGAAGAGTTAAGAGAGGTTTTAAAAAAAGATGAAAA synthetase cloneATCTGCTCTGATAGGTTTTGAACCAAGTGGTAAAATACATTTA #2 nucleotideGGGCATTATCTCCAAATAAAAAAGATGATTGATTTACAAAATG sequenceCTGGATTTGATATAATTATAACTTTGGCTGATTTATCTGCCTATTTAAACCAGAAAGGAGAGTTGGATGAGATTAGAAAAATAGGAGATTATAACAAAAAAGTTTTTGAAGCAATGGGGTTAAAGGCAAAATATGTTTATGGAAGTGAATTCCAGCTTGATAAGGATTATACACTGAATGTCTATAGATTGGCTTTAAAAACTACCTTAAAAAGAGCAAGAAGGAGTATGGAACTTATAGCAAGAGAGGATGAAAATCCAAAGGTTGCTGAAGTTATCTATCCAATAATGGGTGTTAATGTTATTCATTATCATGGCGTTGATGTTGCAGTTGGAGGGATGGAGCAGAGAAAAATACACATGTTAGCAAGGGAGCTTTTACCAAAAAAGGTTGTTTGTATTCACAACCCTGTCTTAACGGGTTTGGATGGAGAAGGAAAGATGAGTTCTTCAAAAGGGAATTTTATAGCTGTTGATGACTCTCCAGAAGAGATTAGGGCTAAGATAAAGAAAGCATACTGCCCAGCTGGAGTTGTTGAAGGAAATCCAATAATGGAGATAGCTAAATACTTCCTTGAATATCCTTTAACCATAAAAAGGCCAGAAAAATTTGGTGGAGATTTGACAGTTAATAGCTATGAGGAGTTAGAGAGTTTATTTAAAAATAAGGAATTGCATCCAATGGATTTAAAAAATGCTGTAGCTGAAGAACTTATAAAGATTTTAGAGCCA ATTAGAAAGAGATTATAA 28biphenylalanine MDEFEMIKRNTSEIISEEELREVLKKDEKSAAIGFEPSGKIHLaminoacyl-tRNA GHYLQIKKMIDLQNAGFDIIISLADLHAYLNQKGELDEIRKIGsynthetase clone DYNKKVFEAMGLKAKYVYGSERELDKDYTLNVYRLALKTTLKR#3 amino acid ARRSMELIAREDENPKVAEVIYPIMQVNSIHYSGVDVAVGGMEsequence (derived QRKIHMLARELLPKKVVCIHNPVLTGLDGEGKMSSSKGNFIAVfrom wild-type DDSPEEIRAKIKKAYCPAGVVEGNPIMEIAKYFLEYPLTIKRP MethanococcusEKFGGDLTVNSYEELESLFKNKELHPMDLKNAVAEELIKILEP jannaschii tyrosyl IRKRLtRNA-synthetase) 29 biphenylalanineATGGACGAATTTGAAATGATAAAGAGAAACACATCTGAAATTA aminoacyl-tRNATCAGCGAGGAAGAGTTAAGAGAGGTTTTAAAAAAAGATGAAAA synthetase cloneATCTGCTGCTATAGGTTTTGAACCAAGTGGTAAAATACATTTA #3 nucleotideGGGCATTATCTCCAAATAAAAAAGATGATTGATTTACAAAATG sequenceCTGGATTTGATATAATTATATCGTTGGCTGATTTACACGCCTATTTAAACCAGAAAGGAGAGTTGGATGAGATTAGAAAAATAGGAGATTATAACAAAAAAGTTTTTGAAGCAATGGGGTTAAAGGCAAAATATGTTTATGGAAGTGAAAGGGAGCTTGATAAGGATTATACACTGAATGTCTATAGATTGGCTTTAAAAACTACCTTAAAAAGAGCAAGAAGGAGTATGGAACTTATAGCAAGAGAGGATGAAAATCCAAAGGTTGCTGAAGTTATCTATCCAATAATGCAGGTTAATAGTATTCATTATAGTGGCGTTGATGTTGCAGTTGGAGGGATGGAGCAGAGAAAAATACACATGTTAGCAAGGGAGCTTTTACCAAAAAAGGTTGTTTGTATTCACAACCCTGTCTTAACGGGTTTGGATGGAGAAGGAAAGATGAGTTCTTCAAAAGGGAATTTTATAGCTGTTGATGACTCTCCAGAAGAGATTAGGGCTAAGATAAAGAAAGCATACTGCCCAGCTGGAGTTGTTGAAGGAAATCCAATAATGGAGATAGCTAAATACTTCCTTGAATATCCTTTAACCATAAAAAGGCCAGAAAAATTTGGTGGAGATTTGACAGTTAATAGCTATGAGGAGTTAGAGAGTTTATTTAAAAATAAGGAATTGCATCCAATGGATTTAAAAAATGCTGTAGCTGAAGAACTTATAAAGATTTTAGAGCCA ATTAGAAAGAGATTATAA 30biphenylalanine MDEFEMIKRNTSEIISEEELREVLKKDEKSAHIGFEPSGKIHLaminoacyl-tRNA GHYLQIKKMIDLQNAGFDIIIVLADLHAYLNQKGELDEIRKIGsynthetase clone DYNKKVFEAMGLKAKYVYGSESKLDKDYTLNVYRLALKTTLKR#4 amino acid ARRSMELIAREDENPKVAEVIYPIMQVNGIHYLGVDVAVGGMEsequence (derived QRKIHMLARELLPKKVVCIHNPVLTGLDGEGKMSSSKGNFIAVfrom wild-type DDSPEEIRAKIKKAYCPAGVVEGNPIMEIAKYFLEYPLTIKRP MethanococcusEKFGGDLTVNSYEELESLFKNKELHPMDLKNAVAEELIKILEP jannaschii tyrosyl IRKRLtRNA-synthetase) 31 biphenylalanineATGGACGAATTTGAAATGATAAAGAGAAACACATCTGAAATTA aminoacyl-tRNATCAGCGAGGAAGAGTTAAGAGAGGTTTTAAAAAAAGATGAAAA synthetase cloneATCTGCTCATATAGGTTTTGAACCAAGTGGTAAAATACATTTA #4 nucleotideGGGCATTATCTCCAAATAAAAAAGATGATTGATTTACAAAATG sequenceCTGGATTTGATATAATTATAGTTTTGGCTGATTTACACGCCTATTTAAACCAGAAAGGAGAGTTGGATGAGATTAGAAAAATAGGAGATTATAACAAAAAAGTTTTTGAAGCAATGGGGTTAAAGGCAAAATATGTTTATGGAAGTGAATCGAAGCTTGATAAGGATTATACACTGAATGTCTATAGATTGGCTTTAAAAACTACCTTAAAAAGAGCAAGAAGGAGTATGGAACTTATAGCAAGAGAGGATGAAAATCCAAAGGTTGCTGAAGTTATCTATCCAATAATGCAGGTTAATGGTATTCATTATCTTGGCGTTGATGTTGCAGTTGGAGGGATGGAGCAGAGAAAAATACACATGTTAGCAAGGGAGCTTTTACCAAAAAAGGTTGTTTGTATTCACAACCCTGTCTTAACGGGTTTGGATGGAGAAGGAAAGATGAGTTCTTCAAAAGGGAATTTTATAGCTGTTGATGACTCTCCAGAAGAGATTAGGGCTAAGATAAAGAAAGCATACTGCCCAGCTGGAGTTGTTGAAGGAAATCCAATAATGGAGATAGCTAAATACTTCCTTGAATATCCTTTAACCATAAAAAGGCCAGAAAAATTTGGTGGAGATTTGACAGTTAATAGCTATGAGGAGTTAGAGAGTTTATTTAAAAATAAGGAATTGCATCCAATGGATTTAAAAAATGCTGTAGCTGAAGAACTTATAAAGATTTTAGAGCCA ATTAGAAAGAGATTATAA 32biphenylalanine MDEFEMIKRNTSEIISEEELREVLKKDEKSAGIGFEPSGKIHLaminoacyl-tRNA GHYLQIKKMIDLQNAGFDIIIVLADLHAYLNQKGELDEIRKIGsynthetase clone DYNKKVFEAMGLKAKYVYGSEADLDKDYTLNVYRLALKTTLKR#5 amino acid ARRSMELIAREDENPKVAEVIYPIMQVNSIHYRGVDVAVGGMEsequence (derived QRKIHMLARELLPKKVVCIHNPVLTGLDGEGKMSSSKGNFIAVfrom wild-type DDSPEEIRAKIKKAYCPAGVVEGNPIMEIAKYFLEYPLTIKRP MethanococcusEKFGGDLTVNSYEELESLFKNKELHPMDLKNAVAEELIKILEP jannaschii tyrosyl IRKRLtRNA-synthetase) 33 biphenylalanineATGGACGAATTTGAAATGATAAAGAGAAACACATCTGAAATTA aminoacyl-tRNATCAGCGAGGAAGAGTTAAGAGAGGTTTTAAAAAAAGATGAAAA synthetase cloneATCTGCTGGGATAGGTTTTGAACCAAGTGGTAAAATACATTTA #5 nucleotideGGGCATTATCTCCAAATAAAAAAGATGATTGATTTACAAAATG sequenceCTGGATTTGATATAATTATAGTTTTGGCTGATTTACACGCCTATTTAAACCAGAAAGGAGAGTTGGATGAGATTAGAAAAATAGGAGATTATAACAAAAAAGTTTTTGAAGCAATGGGGTTAAAGGCAAAATATGTTTATGGAAGTGAAGCGGATCTTGATAAGGATTATACACTGAATGTCTATAGATTGGCTTTAAAAACTACCTTAAAAAGAGCAAGAAGGAGTATGGAACTTATAGCAAGAGAGGATGAAAATCCAAAGGTTGCTGAAGTTATCTATCCAATAATGCAGGTTAATTCGATTCATTATCGTGGCGTTGATGTTGCAGTTGGAGGGATGGAGCAGAGAAAAATACACATGTTAGCAAGGGAGCTTTTACCAAAAAAGGTTGTTTGTATTCACAACCCTGTCTTAACGGGTTTGGATGGAGAAGGAAAGATGAGTTCTTCAAAAGGGAATTTTATAGCTGTTGATGACTCTCCAGAAGAGATTAGGGCTAAGATAAAGAAAGCATACTGCCCAGCTGGAGTTGTTGAAGGAAATCCAATAATGGAGATAGCTAAATACTTCCTTGAATATCCTTTAACCATAAAAAGGCCAGAAAAATTTGGTGGAGATTTGACAGTTAATAGCTATGAGGAGTTAGAGAGTTTATTTAAAAATAAGGAATTGCATCCAATGGATTTAAAAAATGCTGTAGCTGAAGAACTTATAAAGATTTTAGAGCCA ATTAGAAAGAGATTATAA 34biphenylalanine MDEFEMIKRNTSEIISEEELREVLKKDEKSAHIGFEPSGKIHLaminoacyl-tRNA GHYLQIKKNIDLQNAGFDTIIVLADLHAYLNQKGELDEIRKIGsynthetase clone DYNKKVFEAMGLKAKYVYCSERPLDKDYTLNVYRLALKTTLKR#6 amino acid ARRSMELIAREDENPKVAEVIYPIMQVNGIHYLGVDVAVGGMEsequence (derived QRKIHMLARELLPKKVVCIHNPVLTGLDGEGKMSSSKGNFIAVfrom wild-type DDSPEEIRAKIKKAYCPAGVVEGNPIMEIAKYFLEYPLTIKRP MethanococcusEKFGGDLTVNSYEELESLFKNKELHPMDLKNAVAEELIKILEP jannaschii tyrosyl IRKRLtRNA-synthetase) 35 biphenylalanineATGGACGAATTTGAAATGATAAAGAGAAACACATCTGAAATTA aminoacyl-tRNATCAGCGAGGAAGAGTTAAGAGAGGTTTTAAAAAAAGATGAAAA synthetase cloneATCTGCTCATATAGGTTTTGAACCAAGTGGTAAAATACATTTA #6 nucleotideGGGCATTATCTCCAAATAAAAAAGATGATTGATTTACAAAATG sequenceCTGGATTTGATATAATTATAGTTTTGGCTGATTTACACGCCTATTTAAACCAGAAAGGAGAGTTGGATGAGATTAGAAAAATAGGAGATTATAACAAAAAAGTTTTTGAAGCAATGCGGTTAAAGGCAAAATATGTTTATGGAAGTGAAAGGCCTCTTGATAAGGATTATACACTGAATGTCTATAGATTGGCTTTAAAAACTACCTTAAAAAGAGCAAGAAGGAGTATGGAACTTATAGCAAGAGAGGATGAAAATCCAAAGGTTGCTGAAGTTATCTATCCAATAATGCAGGTTAATGGTATTCATTATCTGGGCGTTGATGTTGCAGTTGGAGGGATGGAGCAGAGAAAAATACACATGTTAGCAAGGGAGCTTTTACCAAAAAAGGTTGTTTGTATTCACAACCCTGTCTTAACGGGTTTGGATGGAGAAGGAAAGATGAGTTCTTCAAAAGGGAATTTTATAGCTGTTGATGACTCTCCAGAAGAGATTAGGGCTAAGATAAAGAAAGCATACTGCCCAGCTGGAGTTGTTGAAGGAAATCCAATAATGGAGATAGCTAAATACTTCCTTGAATATCCTTTAACCATAAAAAGGCCAGAAAAATTTGGTGGAGATTTGACAGTTAATAGCTATGAGGAGTTAGAGAGTTTATTTAAAAATAAGGAATTGCATCCAATGGATTTAAAAAATGCTGTAGCTGAAGAACTTATAAAGATTTTAGAGCCA ATTAGAAAGAGATTATAA 36biphenylalanine MDEFEMIKRNTSEIISEEELREVLKKDEKSAHIGFEPSGKIHLaminoacyl-tRNA GHYLQIKKMIDLQNAGFDIIIHLADLHAYLNQKGELDEIRKIGsynthetase clone DYNKKVFEAMGLKAKYVYGSEWMLDKDYTLNVYRLALKTTLKR#7 amino acid ARRSMELIAREDENPKVAEVIYPIMQVNGIHYKGVDVAVGGMEsequence (derived QRKIHMLARELLPKKVVCIHNPVLTGLDGEGKMSSSKGNFIAVfrom wild-type DDSPEEIRAKIKKAYCPAGVVEGNPIMEIAKYFLEYPLTIKRP MethanococcusEKFGGDLTVNSYEELESLFKNKELHPMDLKNAVAEELIKILEP jannaschii tyrosyl IRKRLtRNA-synthetase) 37 biphenylalanineATGGACGAATTTGAAATGATAAAGAGAAACACATCTGAAATTA aminoacyl-tRNATCAGCGAGGAAGAGTTAAGAGAGGTTTTAAAAAAAGATGAAAA synthetase cloneATCTGCTCATATAGGTTTTGAACCAAGTGGTAAAATACATTTA #7 nucleotideGGGCATTATCTCCAAATAAAAAAGATGATTGATTTACAAAATG sequenceCTGGATTTGATATAATTATACATTTGGCTGATTTACACGCCTATTTAAACCAGAAAGGAGAGTTGGATGAGATTAGAAAAATAGGAGATTATAACAAAAAAGTTTTTGAAGCAATGGGGTTAAAGGCAAAATATGTTTATGGAAGTGAATGGATGCTTGATAAGGATTATACACTGAATGTCTATAGATTGGCTTTAAAAACTACCTTAAAAAGAGCAAGAAGGAGTATGGAACTTATAGCAAGAGAGGATGAAAATCCAAAGGTTGCTGAAGTTATCTATCCAATAATGCAGGTTAATGGGATTCATTATAAGGGCGTTGATGTTGCAGTTGGAGGGATGGAGCAGAGAAAAATACACATGTTAGCAAGGGAGCTTTTACCAAAAAAGGTTGTTTGTATTCACAACCCTGTCTTAACGGGTTTGGATGGAGAAGGAAAGATGAGTTCTTCAAAAGGGAATTTTATAGCTGTTGATGACTCTCCAGAAGAGATTAGGGCTAAGATAAAGAAAGCATACTGCCCAGCTGGAGTTGTTGAAGGAAATCCAATAATGGAGATAGCTAAATACTTCCTTGAATATCCTTTAACCATAAAAAGGCCAGAAAAATTTGGTGGAGATTTGACAGTTAATAGCTATGAGGAGTTAGAGAGTTTATTTAAAAATAAGGAATTGCATCCAATGGATTTAAAAAATGCTGTAGCTGAAGAACTTATAAAGATTTTAGAGCCA ATTAGAAAGAGATTATAA 38bipyridylalanine MDEFEMIKRNTSETTSEEELREVLKKDEKSAEIGFEPSGKIHLaminoacyl-tRNA GHYLQIKKMIDLQNAGFDIIIHLADLHAYLNQKGELDEIRKIGsynthetase clone DYNKKVFEAMGLKAKYVYGSEWMLDKDYTLNVYRLALKTTLKR#1 amino acid ARRSMELIAREDENPKVAEVIYPIMQVNGHHYHGVDVAVGGMEsequence (derived QRKIHMLARELLPKKVVCIHNPVLTGLDGEGKMSSSKGNFIAVfrom wild-type DDSPEEIRAKIKKAYCPAGVVEGNPIMEIAKYFLEYPLTIKRP MethanococcusEKFGGDLTVNSYEELESLFKNKELHPMDLKNAVAEELIKILEP jannaschii tyrosyl IRKRLtRNA-synthetase) 39 bipyridylalanineATGGACGAATTTGAAATGATAAAGAGAAACACATCTGAAATTA aminoacyl-tRNATCAGCGAGGAAGAGTTAAGAGAGGTTTTAAAAAAAGATGAAAA synthetase cloneATCTGCTGAGATAGGTTTTGAACCAAGTGGTAAAATACATTTA #1 nucleotideGGGCATTATCTCCAAATAAAAAAGATGATTGATTTACAAAATG sequenceCTGGATTTGATATAATTATACATTTGGCTGATTTACACGCCTATTTAAACCAGAAAGGAGAGTTGGATGAGATTAGAAAAATAGGAGATTATAACAAAAAAGTTTTTGAAGCAATGGGGTTAAAGGCAAAATATGTTTATGGAAGTGAATGGATGCTTGATAAGGATTATACACTGAATGTCTATAGATTGGCTTTAAAAACTACCTTAAAAAGAGCAAGAAGGAGTATGGAACTTATAGCAAGAGAGGATGAAAATCCAAAGGTTGCTGAAGTTATCTATCCAATAATGCAGGTTAATGGTCATCATTATCATGGCGTTGATGTTGCAGTTGGAGGGATGGAGCAGAGAAAAATACACATGTTAGCAAGGGAGCTTTTACCAAAAAAGGTTGTTTGTATTCACAACCCTGTCTTAACGGGTTTGGATGGAGAAGGAAAGATGAGTTCTTCAAAAGGGAATTTTATAGCTGTTGATGACTCTCCAGAAGAGATTAGGGCTAAGATAAAGAAAGCATACTGCCCAGCTGGAGTTGTTGAAGGAAATCCAATAATGGAGATAGCTAAATACTTCCTTGAATATCCTTTAACCATAAAAAGGCCAGAAAAATTTGGTGGAGATTTGACAGTTAATAGCTATGAGGAGTTAGAGAGTTTATTTAAAAATAAGGAATTGCATCCAATGGATTTAAAAAATGCTGTAGCTGAAGAACTTATAAAGATTTTAGAGCCA ATTAGAAAGAGATTATAA 40bipyridylalanine MDEFEMIKRNTSEIISEEELREVLKKDEKSAGIGFEPSGKIHLaminoacyl-tRNA GHYLQIKKMIDLQNAGFDIIIYLADLAAYLNQKGELDEIRKIGsynthetase clone DYNKKVFEAMGLKAKYVYGSEFQLDKDYTLNVYRLALKTTLKR#2 amino acid ARRSMELIAREDENPKVAEVIYPIMEVNGWHYSGVDVAVGGMEsequence (derived QRKIHMLARELLPKKVVCIHNPVLTGLDGEGKMSSSKGNFIAVfrom wild-type DDSPEEIRAKIKKAYCPAGVVEGNPIMEIAKYFLEYPLTIKRP MethanococcusEKFGGDLTVNSYEELESLFKNKELHPMDLKNAVAEELIKILEP jannaschii tyrosyl IRKRLtRNA-synthetase) 41 bipyridylalanineATGGACGAATTTGAAATGATAAAGAGAAACACATCTGAAATTA aminoacyl-tRNATCAGCGAGGAAGAGTTAAGAGAGGTTTTAAAAAAAGATGAAAA synthetase cloneATCTGCTGGTATAGGTTTTGAACCAAGTGGTAAAATACATTTA #2 nucleotideGGGCATTATCTCCAAATAAAAAAGATGATTGATTTACAAAATG sequenceCTGGATTTGATATAATTATATATTTGGCTGATTTAGCTGCCTATTTAAACCAGAAAGGAGAGTTGGATGAGATTAGAAAAATAGGAGATTATAACAAAAAAGTTTTTGAAGCAATGGGGTTAAAGGCAAAATATGTTTATGGAAGTGAATTCCAGCTTGATAAGGATTATACACTGAATGTCTATAGATTGGCTTTAAAAACTACCTTAAAAAGAGCAAGAAGGAGTATGGAACTTATAGCAAGAGAGGATGAAAATCCAAAGGTTGCTGAAGTTATCTATCCAATAATGGAGGTTAATGGTTGGCATTATAGTGGCGTTGATGTTGCAGTTGGAGGGATGGAGCAGAGAAAAATACACATGTTAGCAAGGGAGCTTTTACCAAAAAAGGTTGTTTGTATTCACAACCCTGTCTTAACGGGTTTGGATGGAGAAGGAAAGATGAGTTCTTCAAAAGGGAATTTTATAGCTGTTGATGACTCTCCAGAAGAGATTAGGGCTAAGATAAAGAAAGCATACTGCCCAGCTGGAGTTGTTGAAGGAAATCCAATAATGGAGATAGCTAAATACTTCCTTGAATATCCTTTAACCATAAAAAGGCCAGAAAAATTTGGTGGAGATTTGACAGTTAATAGCTATGAGGAGTTAGAGAGTTTATTTAAAAATAAGGAATTGCATCCAATGGATTTAAAAAATGCTGTAGCTGAAGAACTTATAAAGATTTTAGAGCCA ATTAGAAAGAGATTATAA 421,5-dansylalanine MEEQYRPEEIESKVQLHWDEKRTFEVTEDESKEKYYCLSANPYaminoacyl-tRNA PSGRLHMGHVRNYTIGDVIARYQRMLGKNVLQPIGWDAFGLPAsynthetase clone EGAAVKNNTAPAPWTYDNIAYMKNQLKMLGFGYDWSRELATCTB8 amino acid PEYYRWEQKFFTELYKKGLVYKKTSAVNWCPNDQTVLANEQVIsequence (derived DGCCWRCDTKVERKEIPQWFIKITAYADELLNDLDKLDHWPDTfrom wild-type VKTMQRNWIGRSEGVEITFNVNDYDNTLTVYTTRPDTFMGCTYE. coli leucyl- LAVAAGHPLAQKAAENNPELAAFIDECRNTKVAEAEMATMEKKtRNA synthetase) GVDTGFKAVHPLTGEEIPVWAANFVLMEYGTGAVMAVPGHDQRDYEFASKYGLNIKPVILAADGSEPDLSQQALTEKGVLFNSGEFNGLDHEAAFNAIADKLTAMGVGERKVNYRLRDWGVSRQRYWGAPIPMVTLEDGTVMPTPDDQLPVILPEDVVMDGITSPIKADPEWAKTTVNGMPALRETDTFDTFMESCWIYARYTCPQYKEGMLDSEAANYWLPVDIGIGGIEHAIMTLLYFRFFHKLMRDAGMVNSDEPAKQLLCQGMVLADAFYYVGENGERNWVSPVDAIVERDEKGRIVKAKDAAGHELVYTGMSKMSKSKNNGIDPQVMVERYGADTVRLFNNFASPADMTLEWQESGVEGANRFLKRVWKLVYEHTAKGDVAALNVDALTENQKALRRDVHKTIAKVTDDIGRRQTENTAIAAIMELMNKLAKAPTDGEQDRALMQEALLAVVRMLNPFTPHICFTLWQELKGEGDIDNAPWPVADEKAMVEDSTLVVVQVNGKVRAKITVPVDATEEQVRERAGQEHLVAKYLDGVTVRKVIYVPGKLLNLVVG 43 1,5-dansylalanineATGGAAGAGCAATACCGCCCGGAAGAGATAGAATCCAAAGTAC aminoacyl-tRNAAGCTTCATTGGGATGAGAAGCGCACATTTGAAGTAACCGAAGA synthetase cloneCGAGAGCAAAGAGAAGTATTACTGCCTGTCTGCTAATCCCTAT B8n ucleotideCCTTCTGGTCGACTACACATGGGCCACGTACGTAACTACACCA sequenceTCGGTGACGTGATCGCCCGCTACCAGCGTATGCTGGGCAAAAACGTCCTGCAGCCGATCGGCTGGGACGCGTTTGGTCTGCCTGCGGAAGGCGCGGCGGTGAAAAACAACACCGCTCCGGCACCGTGGACGTACGACAACATCGCGTATATGAAAAACCAGCTCAAAATGCTGGGCTTTGGTTATGACTGGAGCCGCGAGCTGGCAACCTGTACGCCGGAATACTACCGTTGGGAACAGAAATTCTTCACCGAGCTGTATAAAAAAGGCCTGGTATATAAGAAGACTTCTGCGGTCAACTGGTGTCCGAACGACCAGACCGTACTGGCGAACGAACAAGTTATCGACGGCTGCTGCTGGCGCTGCGATACCAAAGTTGAACGTAAAGAGATCCCGCAGTGGTTTATCAAAATCACTGCTTACGCTGACGAGCTGCTCAACGATCTGGATAAACTGGATCACTGGCCAGACACCGTTAAAACCATGCAGCGTAACTGGATCGGTCGTTCCGAAGGCGTGGAGATCACCTTCAACGTTAACGACTATGACAACACGCTGACCGTTTACACTACCCGCCCGGACACCTTTATGGGTTGTACCTACCTGGCGGTAGCTGCGGGTCATCCGCTGGCGCAGAAAGCGGCGGAAAATAATCCTGAACTGGCGGCCTTTATTGACGAATGCCGTAACACCAAAGTTGCCGAAGCTGAAATGGCGACGATGGAGAAAAAAGGCGTCGATACTGGCTTTAAAGCGGTTCACCCATTAACGGGCGAAGAAATTCCCGTTTGGGCAGCAAACTTCGTATTGATGGAGTACGGCACGGGCGCAGTTATGGCGGTACCGGGGCACGACCAGCGCGACTACGAGTTTGCCTCTAAATACGGCCTGAACATCAAACCGGTTATCCTGGCAGCTGACGGCTCTGAGCCAGATCTTTCTCAGCAAGCCCTGACTGAAAAAGGCGTGCTGTTCAACTCTGGCGAGTTCAACGGTCTTGACCATGAAGCGGCCTTCAACGCCATCGCCGATAAACTGACTGCGATGGGCGTTGGCGAGCGTAAAGTGAACTACCGCCTGCGCGACTGGGGTGTTTCCCGTCAGCGTTACTGGGGCGCGCCGATTCCGATGGTGACTCTAGAAGACGGTACCGTAATGCCGACCCCGGACGACCAGCTGCCGGTGATCCTGCCGGAAGATGTGGTAATGGACGGCATTACCAGCCCGATTAAAGCAGATCCGGAGTGGGCGAAAACTACCGTTAACGGTATGCCAGCACTGCGTGAAACCGACACTTTCGACACCTTTATGGAGTCCTGCTGGATTTATGCGCGCTACACTTGCCCGCAGTACAAAGAAGGTATGCTGGATTCCGAAGCGGCTAACTACTGGCTGCCGGTGGATATCGGTATTGGTGGTATTGAACACGCCATTATGACGCTGCTGTACTTCCGCTTCTTCCACAAACTGATGCGTGATGCAGGCATGGTGAACTCTGACGAACCAGCGAAACAGTTGCTGTGTCAGGGTATGGTGCTGGCAGATGCCTTCTACTATGTTGGCGAAAACGGCGAACGTAACTGGGTTTCCCCGGTTGATGCTATCGTTGAACGTGACGAGAAAGGCCGTATCGTGAAAGCGAAAGATGCGGCAGGCCATGAACTGGTTTATACCGGCATGAGCAAAATGTCCAAGTCGAAGAACAACGGTATCGACCCGCAGGTGATGGTTGAACGTTACGGCGCGGACACCGTTCGTCTGTTTATGATGTTTGCTTCTCCGGCTGATATGACTCTCGAATGGCAGGAATCCGGTGTGGAAGGGGCTAACCGCTTCCTGAAACGTGTCTGGAAACTGGTTTACGAGCACACAGCAAAAGGTGATGTTGCGGCACTGAACGTTGATGCGCTGACTGAAAATCAGAAAGCGCTGCGTCGCGATGTGCATAAAACGATCGCTAAAGTGACCGATGATATCGGCCGTCGTCAGACCTTCAACACCGCAATTGCGGCGATTATGGAGCTGATGAACAAACTGGCGAAAGCACCAACCGATGGCGAGCAGGATCGCGCTCTGATGCAGGAAGCACTGCTGGCCGTTGTCCGTATGCTTAACCCGTTCACCCCGCACATCTGCTTCACGCTGTGGCAGGAACTGAAAGGCGAAGGCGATATCGACAACGCGCCGTGGCCGGTTGCTGACGAAAAAGCGATGGTGGAAGACTCCACGCTGGTCGTGGTGCAGGTTAACGGTAAAGTCCGTGCCAAAATCACCGTTCCGGTGGACGCAACGGAAGAACAGGTTCGCGAACGTGCTGGCCAGGAACATCTGGTAGCAAAATATCTTGATGGCGTTACTGTACGTAAAGTGATTTACGTACCAGGTAAACTCCTCAATCTGGTCGTTGGC TAA 44 1,5-dansylalanineMEEQYRPEEIESKVQLHWDEKRTFEVTEDESKEKYYCLSANPY aminoacyl-tRNAPSGRLHMGHVRNYTIGDVIARYQRMLGKNVLQPIGWDAFGLPA synthetase T252AEGAAVKNNTAPAPWTYDNIAYMKNQLKMLGFGYDWSRELATCT amino acid sequencePEYYRWEQKFFTELYKKGLVYKKTSAVNWCPNDQTVLANEQVI (derived from wild-DGCCWRCDTKVERKEIPQWFIKITAYADELLNDLDKLDHWPDT type E. coli leucyl-VKTMQRNWIGRSEGVEITFNVNDYDNTLTVYTTRPDAFMGCTY tRNA synthetase)LAVAAGHPLAQKAAENNPELAAFIDECRNTKVAEAEMATMEKKGVDTGFKAVHPLTGEEIPVWAANFVLMEYGTGAVMAVPGHDQRDYEFASKYGLNIKPVILAADGSEPDLSQQALTEKGVLFNSGEFNGLDHEAAFNAIADKLTAMGVGERKVNYRLRDWGVSRQRYWGAPIPMVTLEDGTVMPTPDDQLPVILPEDVVMDGITSPIKADPEWAKTTVNGMPALRETDTFDTFMESCWIYARYTCPQYKEGMLDSEAANYWLPVDIGIGGIEHAIMTLLYFRFFHKLMRDAGMVNSDEPAKQLLCQGMVLADAFYYVGENGERNWVSPVDAIVERDEKGRIVKAKDAAGHELVYTGMSKNSKSKNNGIDPQVMVERYGADTVRLFMMFASPADMTLEWQESGVEGANRFLKRVWKLVYEHTAKGDVAALNVDALTENQKALRRDVHKTIAKVTDDIGRRQTENTAIAAIMELMNKLAKAPTDGEQDRALMQEALLAVVRMLNPFTPHICFTLWQELKGEGDIDNAPWPVADEKAMVEDSTLVVVQVNGKVRAKITVPVDATEEQVRERAGQEHLVAKYLDGVTVRKVIYVPGKLLNLVVG 45 1,5-dansylalanineATGGAAGAGCAATACCGCCCGGAAGAGATAGAATCCAAAGTAC aminoacyl-tRNAAGCTTCATTGGGATGAGAAGCGCACATTTGAAGTAACCGAAGA synthetase T252ACGAGAGCAAAGAGAAGTATTACTGCCTGTCTGCTAATCCCTAT nucleotide sequenceCCTTCTGGTCGACTACACATGGGCCACGTACGTAACTACACCATCGGTGACGTGATCGCCCGCTACCAGCGTATGCTGGGCAAAAACGTCCTGCAGCCGATCGGCTGGGACGCGTTTGGTCTGCCTGCGGAAGGCGCGGCGGTGAAAAACAACACCGCTCCGGCACCGTGGACGTACGACAACATCGCGTATATGAAAAACCAGCTCAAAATGCTGGGCTTTGGTTATGACTGGAGCCGCGAGCTGGCAACCTGTACGCCGGAATACTACCGTTGGGAACAGAAATTCTTCACCGAGCTGTATAAAAAAGGCCTGGTATATAAGAAGACTTCTGCGGTCAACTGGTGTCCGAACGACCAGACCGTACTGGCGAACGAACAAGTTATCGACGGCTGCTGCTGGCGCTGCGATACCAAAGTTGAACGTAAAGAGATCCCGCAGTGGTTTATCAAAATCACTGCTTACGCTGACGAGCTGCTCAACGATCTGGATAAACTGGATCACTGGCCAGACACCGTTAAAACCATGCAGCGTAACTGGATCGGTCGTTCCGAAGGCGTGGAGATCACCTTCAACGTTAACGACTATGACAACACGCTGACCGTTTACACTACCCGCCCGGACGCGTTTATGGGTTGTACCTACCTGGCGGTAGCTGCGGGTCATCCGCTGGCGCAGAAAGCGGCGGAAAATAATCCTGAACTGGCGGCCTTTATTGACGAATGCCGTAACACCAAAGTTGCCGAAGCTGAAATGCCGACGATGGAGAAAAAAGGCGTCGATACTGGCTTTAAAGCGGTTCACCCATTAACGGGCGAAGAAATTCCCGTTTGGGCAGCAAACTTCGTATTGATGGAGTACGGCACGGGCGCAGTTATGGCGGTACCGGGGCACGACCAGCGCGACTACGAGTTTGCCTCTAAATACGGCCTGAACATCAAACCGGTTATCCTGGCAGCTGACGGCTCTGAGCCAGATCTTTCTCAGCAAGCCCTGACTGAAAAAGGCGTGCTGTTCAACTCTGGCGAGTTCAACGGTCTTGACCATGAAGCGGCCTTCAACGCCATCGCCGATAAACTGACTGCGATGGGCGTTGGCGAGCGTAAAGTGAACTACCGCCTGCGCGACTGGGGTGTTTCCCGTCAGCGTTACTGGGGCGCGCCGATTCCGATGGTGACTCTAGAAGACGGTACCGTAATGCCGACCCCGGACGACCAGCTGCCGGTGATCCTGCCGGAAGATGTGGTAATGGACGGCATTACCAGCCCGATTAAAGCAGATCCGGAGTGGGCGAAAACTACCGTTAACGGTATGCCAGCACTGCGTGAAACCGACACTTTCGACACCTTTATGGAGTCCTGCTGGATTTATGCGCGCTACACTTGCCCGCAGTACAAAGAAGGTATGCTGGATTCCGAAGCGGCTAACTACTGGCTGCCGGTGGATATCGGTATTGGTGGTATTGAACACGCCATTATGACGCTGCTCTACTTCCGCTTCTTCCACAAACTGATGCGTGATGCAGGCATGGTGAACTCTGACGAACCAGCGAAACAGTTGCTGTGTCAGGGTATGGTGCTGGCAGATGCCTTCTACTATGTTGGCGAAAACGGCGAACGTAACTGGGTTTCCCCGGTTGATGCTATCGTTGAACGTGACGAGAAAGGCCGTATCGTGAAAGCGAAAGATGCGGCAGGCCATGAACTGGTTTATACCGGCATGAGCAAAATGTCCAAGTCGAAGAACAACGGTATCGACCCGCAGGTGATGGTTGAACGTTACGGCGCGGACACCGTTCGTCTGTTTATGATGTTTGCTTCTCCGGCTGATATGACTCTCGAATGGCAGGAATCCGGTGTGGAAGGGGCTAACCGCTTCCTGAAACGTGTCTGGAAACTGGTTTACGAGCACACAGCAAAAGGTGATGTTGCGGCACTGAACGTTGATGCGCTGACTGAAAATCAGAAAGCGCTGCGTCGCGATGTGCATAAAACGATCGCTAAAGTGACCGATGATATCGGCCGTCGTCAGACCTTCAACACCGCAATTGCGGCGATTATGGAGCTGATGAACAAACTGGCGAAAGCACCAACCGATGGCGAGCAGGATCGCGCTCTGATGCAGGAAGCACTGCTGGCCGTTGTCCGTATGCTTAACCCGTTCACCCCGCACATCTGCTTCACGCTGTGGCAGGAACTGAAAGGCGAAGGCGATATCGACAACGCGCCGTGGCCGGTTGCTGACGAAAAAGCGATGGTGGAAGACTCCACGCTGGTCGTGGTGCAGGTTAACGGTAAAGTCCGTGCCAAAATCACCGTTCCGGTGGACGCAACGGAAGAACAGGTTCGCGAACGTGCTGGCCAGGAACATCTGGTAGCAAAATATCTTGATGGCGTTACTGTACGTAAAGTGATTTACGTACCAGGTAAACTCCTCAATCTGGTCGTTGGC TAAGCGGCC 461,5-dansylalanine MEEQYRPEEIESKVQLHWDEKRTFEVTEDESKEKYYCLSANPYaminoacyl-tRNA PSGRLHMGHVRNYTIGDVIARYQRMLGKNVLQPIGWDAFGLPAsynthetase V338A EGAAVKNNTAPAPWTYDNIAYMKNQLKMLGFGYDWSRELATCT amino acidPEYYRWEQKFFTELYKKGLVYKKTSAVNWCPNDQTVLANEQVI sequence (derivedDGCCWRCDTKVERKEIPQWFIKITAYADELLNDLDKLDHWPDT from wild-typeVKTMQRNWIGRSEGVEITFNVNDYDNTLTVYTTRPDTFMGCTY E. coli leucyl-LAVAAGHPLAQKAAENNPELAAFIDECRNTKVAEAEMATMEKK tRNA synthetase)GVDTGFKAVHPLTGEEIPVWAANFVLMEYGTGAVMAAPGHDQRDYEFASKYGLNIKPVILAADGSEPDLSQQALTEKGVLFNSGEFNGLDHEAAFNAIADKLTAMGVGERKVNYRLRDWGVSRQRYWGAPIPMVTLEDGTVMPTPDDQLPVILPEDVVMDGITSPIKADPEWAKTTVNGMPALRETDTFDTFMESCWIYARYTCPQYKEGMLDSEAANYWLPVDIGIGGIEHAINTLLYFRFFHKLMRDAGMVNSDEPAKQLLCQGMVLADAFYYVGENGERNWVSPVDAIVERDEKGRIVKAKDAAGHELVYTGMSKMSKSKNNGIDPQVMVERYGADTVRLFMMFASPADMTLEWQESGVEGANRFLKRVWKLVYEHTAKGDVAALNVDALTENQKALRRDVHKTIAKVTDDIGRRQTFNTAIAAIMELMNKLAKAPTDGEQDRALMQEALLAVVRMLNPFTPHICFTLWQELKGEGDIDNAPWPVADEKAMVEDSTLVVVQVNGKVRAKITVPVDATEEQVRERAGQEHLVAKYLDGVTVRKVIYVPGKLLNLVVG 47 1,5-dansylalanineATGGAAGAGCAATACCGCCCGGAAGAGATAGAATCCAAAGTAC aminoacyl-tRNAAGCTTCATTGGGATGAGAAGCGCACATTTGAAGTAACCGAAGA synthetase V338ACGAGAGCAAAGAGAAGTATTACTGCCTGTCTGCTAATCCCTAT nucleotide sequenceCCTTCTGGTCGACTACACATGGGCCACGTACGTAACTACACCATCGGTGACGTGATCGCCCGCTACCAGCGTATGCTGGGCAAAAACGTCCTGCAGCCGATCGGCTGGGACGCGTTTGGTCTGCCTGCGGAAGGCGCGGCGGTGAAAAACAACACCGCTCCGGCACCGTGGACGTACGACAACATCGCGTATATGAAAAACCAGCTCAAAATGCTGGGCTTTGGTTATGACTGGAGCCGCGAGCTGGCAACCTGTACGCCCGAATACTACCGTTGGGAACAGAAATTCTTCACCGAGCTGTATAAAAAAGGCCTGGTATATAAGAAGACTTCTGCGGTCAACTGGTGTCCGAACGACCAGACCGTACTGGCGAACGAACAAGTTATCGACGGCTGCTGCTGGCGCTGCGATACCAAAGTTGAACGTAAAGAGATCCCGCAGTGGTTTATCAAAATCACTGCTTACGCTGACGAGCTGCTCAACGATCTGGATAAACTGGATCACTGGCCAGACACCGTTAAAACCATGCAGCGTAACTGGATCGGTCGTTCCGAAGGCGTGGAGATCACCTTCAACGTTAACGACTATGACAACACGCTGACCGTTTACACTACCCGCCCGGACACCTTTATGGGTTGTACCTACCTGGCGGTAGCTGCGGGTCATCCGCTGGCGCAGAAAGCGGCGGAAAATAATCCTGAACTGGCGGCCTTTATTGACGAATGCCGTAACACCAAAGTTGCCGAAGCTGAAATGGCGACGATGGAGAAAAAAGGCGTCGATACTGGCTTTAAAGCGGTTCACCCATTAACGGGCGAAGAAATTCCCGTTTGGGCAGCAAACTTCGTATTGATGGAGTACGGCACGGGCGCAGTTATGGCGGCGCCGGGGCACGACCAGCGCGACTACGAGTTTGCCTCTAAATACGGCCTGAACATCAAACCGGTTATCCTGGCAGCTGACGGCTCTGAGCCAGATCTTTCTCAGCAAGCCCTGACTGAAAAAGGCGTGCTGTTCAACTCTGGCGAGTTCAACGGTCTTGACCATGAAGCGGCCTTCAACGCCATCGCCGATAAACTGACTGCGATGGGCGTTGGCGAGCGTAAAGTGAACTACCGCCTGCGCGACTGGGGTGTTTCCCGTCAGCGTTACTGGGGCGCGCCGATTCCGATGGTGACTCTAGAAGACGGTACCGTAATGCCGACCCCGGACGACCAGCTGCCGGTGATCCTGCCGGAAGATGTGGTAATGGACGGCATTACCAGCCCGATTAAAGCAGATCCGGAGTGGGCGAAAACTACCGTTAACGGTATGCCAGCACTGCGTGAAACCGACACTTTCGACACCTTTATGGAGTCCTGCTGGATTTATGCGCGCTACACTTGCCCGCAGTACAAAGAAGGTATGCTGGATTCCGAAGCGGCTAACTACTGGCTGCCGGTGGATATCGGTATTGGTGGTATTGAACACGCCATTATGACGCTGCTCTACTTCCGCTTCTTCCACAAACTGATGCGTGATGCAGGCATGGTGAACTCTGACGAACCAGCGAAACAGTTGCTGTGTCAGGGTATGGTGCTGGCAGATGCCTTCTACTATGTTGGCGAAAACGGCGAACGTAACTGGGTTTCCCCGGTTGATGCTATCGTTGAACGTGACGAGAAAGGCCGTATCGTGAAAGCGAAAGATGCGGCAGGCCATGAACTGGTTTATACCGGCATGAGCAAAATGTCCAAGTCGAAGAACAACGGTATCGACCCGCAGGTGATGGTTGAACGTTACGGCGCGGACACCGTTCGTCTGTTTATGATGTTTGCTTCTCCGGCTGATATGACTCTCGAATGGCAGGAATCCGGTGTGGAAGGGGCTAACCGCTTCCTGAAACGTGTCTGGAAACTGGTTTACGAGCACACAGCAAAAGGTGATGTTGCGGCACTGAACGTTGATGCGCTGACTGAAAATCAGAAAGCGCTGCGTCGCGATGTGCATAAAACGATCGCTAAAGTGACCGATGATATCGGCCGTCGTCAGACCTTCAACACCGCAATTGCGGCGATTATGGAGCTGATGAACAAACTGGCGAAAGCACCAACCGATGGCGAGCAGGATCGCGCTCTGATGCAGGAAGCACTGCTGGCCGTTGTCCGTATGCTTAACCCGTTCACCCCGCACATCTGCTTCACGCTGTGGCAGGAACTGAAAGGCGAAGGCGATATCGACAACGCGCCGTGGCCGGTTGCTGACGAAAAAGCGATGGTGGAAGACTCCACGCTGGTCGTGGTGCAGGTTAACGGTAAAGTCCGTGCCAAAATCACCGTTCCGGTGGACGCAACGGAAGAACAGGTTCGCGAACGTGCTGGCCAGGAACATCTGGTAGCAAAATATCTTGATGGCGTTACTGTACGTAAAGTGATTTACGTACCAGGTAAACTCCTCAATCTGGTCGTTGGC TAAGCGGCC 48o-nitrobenzylcysteine MEEQYRPEEIESKVQLHWDEKRTFEVTEDESKEKYYCLSWSPYaminoacyl-tRNA PSGRLHMGHVRNYTIGDVIARYQRMLGKNVLQPIGWDAFGLPAsynthetase clone EGAAVKNNTAPAPWTYDNIAYMKNQLKMLGFGYDWSRELATCT3H11 amino acid PEYYRWEQKFFTELYKKGLVYKKTSAVNWCPNDQTVLANEQVIsequence (derived DGCCWRCDTKVERKEIPQWFIKITAYADELLNDLDKLDHWPDTfrom wild-type VKTMQRNWIGRSEGVEITFNVNDYDNTLTVYTTRPDTFMGCTYE. coli leucyl- LAVAAGHPLAQKAAENNPELAAFIDECRNTKVAEAEMATMEKKtRNA synthetase) GVDTGFKAVHPLTGEEIPVWAANFVLMEYGTGAVMAVPGHDQRDYEFASKYGLNIKPVILAADGSEPDLSQQALTEKGVLFNSGEFNGLDHEAAFNAIADKLTAMGVGERKVNYRLRDWGVSRQRYWGAPIPMVTLEDGTVMPTPDDQLPVILPEDVVMDGITSPIKADPEWAKTTVNGMPALRETDTFDTFMESCWIYARYTCPQYKEGMLDSEAANYWLPVDIAIGGIEHAIMGLLYFRFFHKLMRDAGMVNSDEPAKQLLCQGMVLADAFYYVGENGERNWVSPVDAIVERDEKGRIVKAKDAAGHELVYTGMSKMSKSKNNGIDPQVMVERYGADTVRLFMMFASPADMTLEWQESGVEGANRFLKRVWKLVYEHTAKGDVAALNVDALTENQKALRRDVHKTIAKVTDDIGRRQTFNTAIAAIMELMNKLAKAPTDGEQDRALMQEALLAVVRMLNPFTPHICFTLWQELKGEGDIDNAPWPVADEKAMVEDSTLVVVQVNGKVRAKITVPVDATEEQVRERAGQEHLVAKYLDGVTVRKVIYVPGKLLNLVVG 49 o-nitrobenzylcysteineATGGAAGAGCAATACCGCCCGGAAGAGATAGAATCCAAAGTAC aminoacyl-tRNAAGCTTCATTGGGATGAGAAGCGCACATTTGAAGTAACCGAAGA synthetase cloneCGAGAGCAAAGAGAAGTATTACTGCCTGTCTTGGTCGCCCTAT 3H11 nucleotideCCTTCTGGTCGACTACACATGGGCCACGTACGTAACTACACCA sequenceTCGGTGACGTGATCGCCCGCTACCAGCGTATGCTGGGCAAAAACGTCCTGCAGCCGATCGGCTGGGACGCGTTTGGTCTGCCTGCGGAAGGCGCGGCGGTGAAAAACAACACCGCTCCGGCACCGTGGACGTACGACAACATCGCGTATATGAAAAACCAGCTCAAAATGCTGGGCTTTGGTTATGACTGGAGCCGCGAGCTGGCAACCTGTACGCCGGAATACTACCGTTGGGAACAGAAATTCTTCACCGAGCTGTATAAAAAAGGCCTGGTATATAAGAAGACTTCTGCGGTCAACTGGTGCCCGAACGACCAGACCGTACTGGCGAACGAACAAGTTATCGACGGCTGCTGCTGGCGCTGCGATACCAAAGTTGAACGTAAAGAGATCCCGCAGTGGTTTATCAAAATCACTGCTTACGCTGACGAGCTGCTCAACGATCTGGATAAACTGGATCACTGGCCAGACACCGTTAAAACCATGCAGCGTAACTGGATCGGTCGTTCCGAAGGCGTGGAGATCACCTTCAACGTTAACGACTATGACAACACGCTGACCGTTTACACTACCCGCCCGGACACCTTTATGGGTTGTACCTACCTGGCGGTAGCTGCCGGTCATCCGCTGGCGCAGAAAGCGGCGGAAAATAATCCTGAACTGGCGGCCTTTATTGACGAATGCCGTAACACCAAAGTTGCCGAAGCTGAAATGGCGACGATGGAGAAAAAAGGCGTCGATACTGGCTTTAAAGCGGTTCACCCATTAACGGGCGAAGAAATTCCCGTTTGGGCAGCAAACTTCGTATTGATGGAGTACGGCACGGGCGCAGTTATGGCGGTACCGGGGCACGACCAGCGCGACTACGAGTTTGCCTCTAAATACGGCCTGAACATCAAACCGGTTATCCTGGCAGCTGACGGCTCTGAGCCAGATCTTTCTCAGCAAGCCCTGACTGAAAAAGGCGTGCTGTTCAACTCTGGCGAGTTCAACGGTCTTGACCATGAAGCGGCCTTCAACGCCATCGCCGATAAACTGACTGCGATGGGCGTTGGCGAGCGTAAAGTGAACTACCGCCTGCGCGACTGGGGTGTTTCCCGTCAGCGTTACTGGGGCGCGCCGATTCCGATGGTGACGCTGGAAGACGGTACCGTAATGCCGACCCCGGACGACCAGCTGCCGGTGATCCTGCCGGAAGATGTGGTAATGGACGGCATTACCAGCCCGATTAAAGCAGATCCGGAGTGGGCGAAAACTACCGTTAACGGTATGCCAGCACTGCGTGAAACCGACACTTTCGACACCTTTATGGAGTCCTGCTGGATTTATGCGCGCTACACTTGCCCGCAGTACAAAGAAGGTATGCTGGATTCCGAAGCGGCTAACTACTGGCTGCCGGTGGATATCGCGATTGGTGGTATTGAACACGCCATTATGGGGCTGCTCTACTTCCGCTTCTTCCACAAACTGATGCGTGATGCAGGCATGGTGAACTCTGACGAACCAGCGAAACAGTTGCTGTGTCAGGGTATGGTGCTGGCAGATGCCTTCTACTATGTTGGCGAAAACGGCGAACGTAACTGGGTTTCCCCGGTTGATGCTATCGTTGAACGTGACGAGAAAGGCCGTATCGTGAAAGCGAAAGATGCGGCAGGCCATGAACTGGTTTATACCGGCATGAGCAAAATGTCCAAGTCGAAGAACAACGGTATCGACCCGCAGGTGATGGTTGAACGTTACGGCGCGGACACCGTTCGTCTGTTTATGATGTTTGCTTCTCCGGCTGATATGACTCTCGAATGGCAGGAATCCGGTGTGGAAGGGGCTAACCGCTTCCTGAAACGTGTCTGGAAACTGGTTTACGAGCACACAGCAAAAGGTGATGTTGCGGCACTGAACGTTGATGCGCTGACTGAAAATCAGAAAGCGCTGCGTCGCGATGTGCATAAAACGATCGCTAAAGTGACCGATGATATCGGCCGTCGTCAGACCTTCAACACCGCAATTGCGGCGATTATGGAGCTGATGAACAAACTGGCGAAAGCACCAACCGATGGCGAGCAGGACCGCGCTCTGATGCAGGAAGCACTGCTGGCCGTTGTCCGTATGCTTAACCCGTTCACCCCGCACATCTGCTTCACGCTGTGGCAGGAACTGAAAGGCGAAGGCGATATCGACAACGCGCCGTGGCCGGTTGCTGACGAAAAAGCGATGGTGGAAGACTCCACGCTGGTCGTGGTGCAGGTTAACGGTAAAGTCCGTGCCAAAATCACCGTTCCGGTGGACGCAACGGAAGAACAGGTTCGCGAACGTGCTGGCCAGGAACATCTGGTAGCAAAATATCTTGATGGCGTTACTGTACGTAAAGTGATTTACGTACCAGGTAAACTCCTCAATCTGGTCGTTGGC TAA 50 o-nitrobenzylserineMEEQYRPEEIESKVQLHWDEKRTFEVTEDEGKEKYYCLSWSPY aminoacyl-tRNAPSGRLHMGHVRNYTIGDVIARYQRMLGKNVLQPIGWDAFGLPA synthetase cloneEGAAVKNNTAPAPWTYDNIAYMKNQLKMLGFGYDWSRELATCT G2-6 amino acidPEYYRWEQKFFTELYKKGLVYKKTSAVNWCPNDQTVLANEQVI sequence (derivedDGCCWRCDTKVERKEIPQWFIKITAYADELLNDLDKLDHWPDT from wild-typeVKTMQRNWIGRSEGVEITFNVNDYDNTLTVYASRPDTFMGCTY E. coli leucyl-LAVAAGHPLAQKAAENNPELAAFIDECRNTKVAEAEMATMEKK tRNA synthetase)GVDTGFKAVHPLTGEEIPVWAANFVLMEYGTGAVMAVPGHDQRDYEFASKYGLNIKPVILAADGSEPDLSQQALTEKGVLFNSGEFNGLDHEAAFNAIADKLTAMGVGERKVNYRLRDWGVSRQRYWGAPIPMVTLEDGTVMPTPDDQLPVILPEDVVMDGITSPIKADPEWAKTTVNGMPALRETDTFDTFMESCWIYARYTCPQYKEGMLDSEAANYWLPVDIAIGGIEHAIMGLLYFRFFHKLMRDAGMVNSDEPAKQLLCQGMVLADAFYYVGENGERNWVSPVDAIVERDEKGRIVKAKDAAGHELVYTGISKMSKSKNNGIDPQVMVERYGADTVRLFMMFASPADMTLEWQESGVEGANRFLKRAWKLVYEHTAKGDVAALNVDALTENQKALRRDVHKTIAKVTDDIGRRQTFNTAIAAIMELMNKLAKAPTDGEQDRALMQEALLAVVRMLNPFTPHICFTLWQELKGEGDIDNAPWPVADEKAMVEDSTLVVVQVNGKVRAKITVPVDATEEQVRERAGQEHLVAKYLDGVTVRKVIYVPGKLLNLVVG 51 o-nitrobenzylserineATCTCGAAGCACACGAAACTTTTTCCTTCCTTCATTCACGCAC aminoacyl-tRNAACTACTCTCTAATGAGCAACGGTATACGGCCTTCCTTCCAGTT synthetase cloneACTTGAATTTGAAATAAAAAAAAGTTTGCTGTCTTGCTATCAA G2-6 nucleotideGTATAAATAGACCTGCAATTATTAATCTTTTGTTTCCTCGTCA sequenceTTGTTCTCOPTCCCTTTCTTCCTTGTTTCTTTTTCTGCACAATATTTCAAGCTATACCAAGCATACAATCAACTGAATTCAGTATGGAAGAGCAATACCGCCCGGAAGAGATAGAATCCAAAGTACAGCTTCATTGGGATGAGAAGCGCACATTTGAAGTAACCGAAGACGAGGGCAAAGAGAAGTATTACTGCCTGTCTTGGTCGCCCTATCCTTCTGGTCGACTACACATGGGCCACGTACGTAACTACACCATCGGTGACGTGATCGCCCGCTACCAGCGTATGCTGGGCAAAAACGTCCTGCAGCCGATCGGCTGGGACGCGTTTGGTCTGCCTGCGGAAGGCGCGGCGGTGAAAAACAACACCGCTCCGGCACCGTGGACGTACGACAACATCGCGTATATGAAAAACCAGCTCAAAATGCTGGGCTTTGGTTATGACTGGAGCCGCGAGCTGGCAACCTGTACGCCGGAATACTACCGTTGGGAACAGAAATTCTTCACCGAGCTGTATAAAAAAGGCCTGGTATATAAGAAGACTTCTGCGGTCAACTGGTGTCCGAACGACCAGACCGTACTGGCGAACGAACAAGTTATCGACGGCTGCTGCTGGCGCTGCGATACCAAAGTTGAACGTAAAGAGATCCCGCAGTGGTTTATCAAAATCACTGCTTACGCTGACGAGCTGCTCAACGATCTGGATAAACTGGATCACTGGCCAGACACCGTTAAAACCATGCAGCGTAACTGGATCGGTCGTTCCGAAGGCGTGGAGATCACCTTCAACGTTAACGACTATGACAACACGCTGACCGTTTACGCTTCCCGCCCGGACACCTTTATGGGTTGTACCTACCTGGCGGTAGCTGCGGGTCATCCGCTGGCGCAGAAAGCGGCGGAAAATAATCCTGAACTGGCGGCCTTTATTGACGAATGCCGTAACACCAAAGTTGCCGAAGCTGAAATGGCGACGATGGAGAAAAAAGGCGTCGATACTGGCTTTAAAGCGGTTCACCCATTAACGGGCGAAGAAATTCCCGTTTGGGCAGCAAACTTCGTATTGATGGAGTACGGCACGGGCGCAGTTATGGCGGTACCGGGGCACGACCAGCGCGACTACGAGTTTGCCTCTAAATACGGCCTGAACATCAAACCGGTTATCCTGGCAGCTGACGGCTCTGAGCCAGATCTTTCTCAGCAAGCCCTGACTGAAAAAGGCGTGCTGTTCAACTCTGGCGAGTTCAACGGTCTTGACCATGAAGCGGCCTTCAACGCCATCGCCGATAAACTGACTGCGATGGGCGTTGGCGAGCGTAAAGTGAACTACCGCCTGCGCGACTGGGGTGTTTCCCGTCAGCGTTACTGGGGCGCGCCGATTCCGATGGTGACTCTAGAAGACGGTACCGTAATGCCGACCCCGGACGACCAGCTGCCGGTGATCCTGCCGGAAGATGTGGTAATGGACGGCATTACCAGCCCGATTAAAGCAGATCCGGAGTGGGCGAAAACTACCGTTAACGGTATGCCAGCACTGCGTGAAACCGACACTTTCGACACCTTTATGGAGTCCTGCTGGATTTATGCGCGCTACACTTGCCCGCAGTACAAAGAAGGTATGCTGGATTCCGAAGCGGCTAACTACTGGCTGCCGGTGGATATCGCGATTGGTGGTATTGAACACGCCATTATGGGGCTGCTCTACTTCCGCTTCTTCCACAAACTGATGCGTGATGCAGGCATGGTGAACTCTGACGAACCAGCGAAACAGTTGCTGTGTCAGGGTATGGTGCTGGCAGATGCCTTCTACTATGTTGGCGAAAACGGCGAACGTAACTGGGTTTCCCCGGTTGATGCTATCGTTGAACGTGACGAGAAAGGCCGTATCGTGAAAGCGAAAGATGCGGCAGGCCATGAACTGGTTTATACCGGCATAAGCAAAATGTCCAAGTCGAAGAACAACGGTATCGACCCGCAGGTGATGGTTGAACGTTACGGCGCGGACACCGTTCGTCTGTTTATGATGTTTGCTTCTCCGGCTGATATGACTCTCGAATGGCAGGAATCCGGTGTGGAAGGGGCTAACCGCTTCCTGAAACGTGCCTGGAAACTGGTTTACGAGCACACAGCAAAAGGTGATGTTGCGGCACTGAACGTTGATGCGCTGACTGAAAATCAGAAAGCGCTGCGTCGCGATGTGCATAAAACGATCGCTAAAGTGACCGATGATATCGGCCGTCGTCAGACCTTCAACACCGCAATTGCGGCGATTATGGAGCTGATGAACAAACTGGCGAAAGCACCAACCGATGGCGAGCAGGATCGCGCTCTGATGCAGGAAGCACTGCTGGCCGTTGTCCGTATGCTTAACCCGTTCACCCCGCACATCTGCTTCACGCTGTGGCAGGAACTGAAAGGCGAAGGCGATATCGACAACGCGCCGTGGCCGGTTGCTGACGAAAAAGCGATGGTGGAAGACTCCACGCTGGTCGTGGTGCAGGTTAACGGTAAAGTCCGTGCCAAAATCACCGTTCCGGTGGACGCAACGGAAGAACAGGTTCGCGAACGTGCTGGCCAGGAACATCTGGTAGCAAAATATCTTGATGGCGTTACTGTACGTAAAGTGATTTACGTACCAGGTAAACTCCTCAATCTGGTCGTTGGCTAA GCGGCC 52 O-(2-nitrobenzyl)-MDEFEMIKRNTSEIISEEELREVLKKDEKSAGIGFEPSGKIHL L-tyrosineGHYLQIKKMIDLQNAGFDIIIGLADLHAYLNQKGELDEIRKIG aminoacyl-tRNADYNKKVFEAMGLKAKYVYGSEARLDKDYTLNVYRLALKTTLK synthetase cloneRARRSMELIAREDENPKVAEVIYPIMQVNEIHYYGVDVAVGG ONBY-1 amino acidMEQRKIHMLARELLPKKVVCIHNPVLTGLDGEGKMSSSKGNFI sequence (derivedAVDDSPEEIRAKIKKAYCPAGVVEGNPIMEIAKYFLEYPLTIK from wild-typeRPEKFGGDLTVNSYEELESLFKNKELHPMDLKNAVAEELIKIL Methanococcus EPIRKRLjannaschii tyrosyl tRNA-synthetase) 53 O-(2-nitrobenzyl)-MDEFEMIKRNTSEIISEEELREVLKKDEKSAAIGFEPSGKIHL L-tyrosineGHYLQIKKMIDLQNAGFDIIIGLADLHAYLNQKGELDEIRKIG aminoacyl-tRNADYNKKVFEAMGLKAKYVYGSECDLDKDYTLNVYRLALKTTLK synthetase cloneRARRSMELIAREDENPKVAEVIYPIMQVNAIHYGGVDVAVGG ONBY-2 amino acidMEQRKIHMLARELLPKKVVCIHNPVLTGLDGEGKMSSSKGNFI sequence (derivedAVDDSPEEIRAKIKKAYCPAGVVEGNPIMEIAKYFLEYPLTIK from wild-typeRPEKFGGDLTVNSYEELESLFKNKELHPMDLKNAVAEELIKIL Methanococcus EPIRKRLjannaschii tyrosyl tRNA-synthetase) 54 O-(2-nitrobenzyl)-MDEFEMIKRNTSEIISEEELREVLKKDEKSAGIGFEPSGKIHL L-tyrosineGHYLQIKKMIDLQNAGFDIIIGLADLHAYLNQKGELDEIRKIG aminoacyl-tRNADYNKKVFEAMGLKAKYVYGSEEQLDKDYTLNVYRLALKTTLKR synthetase cloneARRSMELIAREDENPKVAEVIYPIMQVNSIHYEGVDVAVGGM ONBY-3 amino acidEQRKIHMLARELLPKKVVCIHNPVLTGLDGEGKMSSSKGNFIA sequence (derivedVDDSPEETRAKIKKAYCPAGVVEGNPIMEIAKYFLEYPLTIKR from wild-typePEKFGGDLTVNSYEELESLFKNKELHPMDLKNAVAEELIKILE Methanococcus PIRKRLjannaschii tyrosyl tRNA-synthetase) 55 p-cyanophenylalanineMDEFEMIKRNTSEIISEEELREVLKKDEKSALIGFEPSGKIHL aminoacyl-tRNAGHYLQIKKMIDLQNAGFDIIIVLADLHAYLNQKGELDEIRKIG synthetase amino acidDYNKKVFEAMGLKAKYVYGSEWMLDKDYTLNVYRLALKTTLKR sequence (derivedARRSMELIAREDENPKVAEVIYPIMQVNGAHYLGVDVAVGGME from wild-typeQRKIHMLARELLPKKVVCIHNPVLTGLDGEGKMSSSKGNFIAV MethanococcusDDSPEEIRAKIKKAYCPAGVVEGNPIMEIAKYFLEYPLTIKRP jannaschii tyrosylEKEGGDLTVNSYEELESLEKNKELHPMDLKNAVAEELIKILEP tRNA-synthetase), IRKRLhaving the substitutions: Tyr32Leu, Leu65Val, Phe108Trp, Gln109Met,Asp158Gly, Ile159Ala 56 p-cyanophenylalanineATGGACGAATTTGAAATGATAAAGAGAAACACATCTGAAATTA aminoacyl-tRNATCAGCGAGGAAGAGTTAAGAGAGGTTTTAAAAAAAGATGAAAA synthetase nucleotideATCTGCTCTGATAGGTTTTGAACCAAGTGGTAAAATACATTTA sequenceGGGCATTATCTCCAAATAAAAAAGATGATTGATTTACAAAATGCTGGATTTGATATAATTATAGTTTTCGCTGATTTACATGCCTATTTAAACCAGAAAGGAGAGTTGGATGAGATTAGAAAAATAGGAGATTATAACAAAAAAGTTTTTGAAGCAATGGGGTTAAAGGCAAAATATGTTTATGGAAGTGAATGGATGCTTGATAAGGATTATACACTGAATGTCTATAGATTGGCTTTAAAAACTACCTTAAAAAGAGCAAGAAGGAGTATGGAACTTATAGCAAGAGAGGATGAAAATCCAAAGGTTGCTGAAGTTATCTATCCAATAATGCAGGTTAATGGTGCTCATTATCTTGGCGTTGATGTTGCAGTTGGGGGGATGGAGCAGAGAAAAATACACATGTTAGCAAGGGAGCTTTTACCAAAAAAGGTTGTTTGTATTCACAACCCTGTCTTAACGGGTTTGGATGGAGAAGGAAAGATGAGTTCTTCAAAAGGGAATTTTATAGCTGTTGATGACTCTCCAGAAGAGATTAGGGCTAAGATAAAGAAAGCATACTGCCCAGCTGGAGTTGTTGAAGGAAATCCAATAATGGAGATAGCTAAATACTTCCTTGAATATCCTTTAACCATAAAAAGGCCAGAAAAATTTGGTGGAGATTTGACAGTTAATAGCTATGAGGAGTTAGAGAGTTTATTTAAAAATAAGGAATTGCATCCAATGGATTTAAAAAATGCTGTAGCTGAAGAACTTATAAAGATTTTAGAGCCA ATTAGAAAGAGATTATAA 57m-cyanophenylalanine MDEFEMIKRNTSEIISEEELREVLKKDEKSAHIGFEPSGKIHLaminoacyl-tRNA GHYLQIKKMIDLQNAGFDIIILLADLSAYLNQKGELDEIRKIGsynthetase amino DYNKKVFEAMGLKAKYVYGSEFQLDKDYTLNVYRLALKTTLKRacid sequence ARRSMELIAREDENPKVAEVIYPIMQVNSSHYPGVDVAVGGME (derived fromQRKIHMLARELLPKKVVCIHNPVLTGLDGEGKMSSSKGNFIAV wild-typeDDSPEEIRAKIKKAYCPAGVVEGNPIMEIAKYFLEYPLTIKRP MethanococcusEKFGGDLTVNSYEELESLFKNKELHPMDLKNAVAEELIKILEP jannaschii tyrosyl IRKRLtRNA-synthetase), having the substitutions: Tyr32His, His70Ser,Asp158Ser, Ile159Ser, Leu162Pro 58 m-cyanophenylalanineATGGACGAATTTGAAATGATAAAGAGAAACACATCTGAAATTA aminoacyl-tRNATCAGCGAGGAAGAGTTAAGAGAGGTTTTAAAAAAAGATGAAAA synthetase nucleotideATCTGCTCATATAGGTTTTGAACCAAGTGGTAAAATACATTTA sequenceGGGCATTATCTCCAAATAAAAAAGATGATTGATTTACAAAATGCTGGATTTGATATAATTATATTGTTGGCTGATTTATCTGCCTATTTAAACCAGAAAGGAGAGTTGGATGAGATTAGAAAAATAGGAGATTATAACAAAAAAGTTTTTGAAGCAATGGGGTTAAAGGCAAAATATGTTTATGGAAGTGAATTCCAGCTTGATAAGGATTATACACTGAATGTCTATAGATTGGCTTTAAAAACTACCTTAAAAAGAGCAAGAAGGAGTATGGAACTTATAGCAAGAGAGGATGAAAATCCAAAGGTTGCTGAAGTTATCTATCCAATAATGCAGGTTAATAGTTCGCATTATCCTGGCGTTGATGTTGCAGTTGGAGGGATGGAGCAGAGAAAAATACACATGTTAGCAAGGGAGCTTTTACCAAAAAAGGTTGTTTGTATTCACAACCCTGTCTTAACGGGTTTGGATGGAGAAGGAAAGATGAGTTCTTCAAAAGGGAATTTTATAGCTGTTGATGACTCTCCAGAAGAGATTAGGGCTAAGATAAAGAAAGCATACTGCCCAGCTGGAGTTGTTGAAGGAAATCCAATAATGGAGATAGCTAAATACTTCCTTGAATATCCTTTAACCATAAAAAGGCCAGAAAAATTTGGTGGAGATTTGACAGTTAATAGCTATGAGGAGTTAGAGAGTTTATTTAAAAATAAGGAATTGCATCCAATGGATTTAAAAAATGCTGTAGCTGAAGAACTTATAAAGATTTTAGAGCCA ATTAGAAAGAGATTATAA 59p-(2-amino-l- MDEFEMIKRNTSEIISEEELREVLKKDEKSADIGFEPSGKIHLhydroxyethyl)-L- GHYLQIKKMIDLQNAGFDIIIELADLHAYLNQKGELDEIRKIGphenylalanine DYNKKVFEAMGLKAKYVYGSERQLDKDYTLNVYRLALKTTLKR aminoacyl-tRNAARRSMELIAREDENPKVAEVIYPIMQVNGIHYNGVDVAVGGME synthetase aminoQRKIHMLARELLPKKVVCIHNPVLTGLDGEGKMSSSKGNFIAV acid sequenceDDSPEEIRAKIKKAYCPAGVVEGNPIMEIAKYFLEYPLTIKRP (derived fromEKFGGDLTVNSYEELESLFKNKELHPMDLKNAVAEELIKILEP wild-type IRKRLMethanococcus jannaschii tyrosyl tRNA-synthetase), having thesubstitutions: Tyr32Asp, Leu65Glu, Phe108Arg, Asp158Gly, Leu162Asn 60p-ethylthiocarbonyl- MDEFEMIKRNTSEIISEEELREVLKKDEKSAAIGFEPSGKIHLL-phenylalanine GHYLQIKKMIDLQNAGFDIIIFLADLHAYLNQKGELDEIRKIGaminoacyl-tRNA DYNKKVFEAMGLKAKYVYGSEWSLDKDYTLNVYRLALKTTLKRsynthetase amino ARRSMELIAREDENPKVAEVIYPIMQVNSIHYHGVDVAVGGMEacid sequence QRKIHMLARELLPKKVVCIHNPVLTGLDGEGKMSSSKGNFIAV (derived fromDDSPEEIRAKIKKAYCPAGVVEGNPIMEIAKYFLEYPLTIKRP wild-typeEKFGGDLTVNSYEELESLFKNKELHPMDLKNAVAEELIKILEP Methanococcus IRKRLjannaschii tyrosyl  tRNA-synthetase), having the substitutions:Tyr32Ala, Leu65Phe, Phe108Trp, Gln109Ser, Asp158Ser, Leu162His 61p-(3-oxobutanoyl)- MDEFEMIKRNTSEIISEEELREVLKKDEKSAGIGFEPSGKIHLL-phenylalanine GHYLQIKKMIDLQNAGFDIIIVLADLHAYLNQKGELDEIRKIGaminoacyl-tRNA DYNKKVFEAMGLKAKYVYGSETQLDKDYTLNVYRLALKTTLKRsynthetase amino ARRSMELIAREDENPKVAEVIYPIMQVNGIHYSGVDVAVGGMEacid sequence QRKIHMLARELLPKKVVCIHNPVLTGLDGEGKMSSSKGNFIAV (derived fromDDSPEEIRAKIKKAYCPAGVVEGNPIMEIAKYFLEYPLTIKRP wild-typeEKFGGDLTVNSYEELESLFKNKELHPMDLKNAVAEELIKILEP Methanococcus IRKRLjannaschii tyrosyl tRNA-synthetase), having the substitutions:Tyr32Gly, Leu65Val, Phe108Thr, Asp158Gly, Leu162Ser 62p-isopropylthiocarbonyl- MDEFEMIKRNTSEIISEEELREVLKKDEKSAGIGFEPSGKIHLL-phenylalanine GHYLQIKKMIDLQNAGFDIIICLADLHAYLNQKGELDEIRKIGaminoacyl-tRNA DYNKKVFEAMGLKAKYVYGSECMLDKDYTLNVYRLALKTTLKRsynthetase amino acid ARRSMELIAREDENPKVAEVIYPIMQVNGIHYYGVDVAVGGMEsequence (derived QRKIHMLARELLPKKVVCIHNPVLTGLDGEGKMSSSKGNFIAVfrom wild-type DDSPEEIRAKIKKAYCPAGVVEGNPIMEIAKYFLEYPLTIKRP MethanococcusEKFGGDLTVNSYEELESLFKNKELHPMDLKNAVAEELIKILEP jannaschii tyrosyl IRKRLtRNA-synthetase), having the substitutions: Tyr32Gly, Leu65Cys,Phe108Cys, Gln109Met, Asp158Gly, Leu162Tyr 63 7-amino-coumarinMDEFEMIKRNTSEIISEEELREVLKKDEKSARIGFEPSGKIHL alanine and 7-GHYLQIKKMIDLQNAGFDIIIALADLMAYLNQKGELDEIRKIG hydroxy-coumarinDYNKKVFEAMGLKAKYVYGSEFQLDKDYTLNVYRLALKTTLKR alanine aminoacyl-ARRSMELIAREDENPKVAEVIYPIMQVNNIHYTGVDVAVGGME tRNA synthetaseQRKIHMLARELLPKKVVCIHNPVLTGLDGEGKMSSSKGNFIAV amino acidDDSPEEIRAKIKKAYCPAGVVEGNPIMEIAKYFLEYPLTIKRP sequence (derivedEKFGGDLTVNSYEELESLFKNKELHPMDLKNAVAEELIKILEP from wild-type IRKRLMethanococcus jannaschii tyrosyl tRNA-synthetase), having thesubstitutions: Y32R, L65A, H70M, D158N and L162T

What is claimed is:
 1. A translation system comprising: (a) a firstunnatural amino acid which is p-(2amino-1-hydroxyethyl)-L-phenylalanine; (b) a first orthogonalaminoacyl-tRNA synthetase (O-RS); and (c) a first orthogonal tRNA(O-tRNA); wherein said first O-RS preferentially aminoacylates saidfirst O-tRNA with said first unnatural amino acid; and wherein the firstO-RS comprises at least 90% identity to the amino acid sequence of SEQID NO
 59. 2. The translation system of claim 1, wherein said first O-RSis derived from a wild-type Methanococcus jannaschii tyrosyl-tRNAsynthetase.
 3. The translation system of claim 1, wherein said firstO-tRNA is an amber suppressor tRNA.
 4. The translation system of claim1, wherein said first O-tRNA comprises or is encoded by a polynucleotidesequence set forth in SEQ ID NO: 1 or
 2. 5. The translation system ofclaim 1, further comprising a nucleic acid encoding a protein ofinterest, said nucleic acid comprising at least one selector codon,wherein said selector codon is recognized by said first O-tRNA.
 6. Thetranslation system of claim 5, further comprising a second O-RS and asecond O-tRNA, wherein the second O-RS preferentially aminoacylates thesecond O-tRNA with a second unnatural amino acid that is different fromthe first unnatural amino acid, and wherein the second O-tRNA recognizesa selector codon that is different from the selector codon recognized bythe first O-tRNA.
 7. The translation system of claim 1, wherein saidsystem comprises a host cell comprising said first unnatural amino acid,said first O-RS and said first O-tRNA.
 8. The translation system ofclaim 7, wherein said host cell is selected from a eubacterial cell anda yeast cell.
 9. The translation system of claim 8, wherein saideubacterial cell is an E. coli cell.
 10. The translation system of claim7, wherein said host cell comprises a polynucleotide encoding said firstO-RS.
 11. The translation system of claim 7, wherein said host cellcomprises a polynucleotide encoding said first O-tRNA.
 12. A method forproducing in a translation system a protein comprising an unnaturalamino acid at a selected position, the method comprising: (a) providinga translation system comprising: (i) a first unnatural amino acid whichis p-(2-amino-1-hydroxyethyl)-L-phenylalanine; (ii) a first orthogonalaminoacyl-tRNA synthetase (O-RS); (iii) a first orthogonal tRNA(O-tRNA), wherein said first O-RS preferentially aminoacylates saidfirst O-tRNA with said unnatural amino acid, wherein said first O-RScomprises an amino acid sequence with at least 90% identity to the aminoacid sequence of SEQ ID NO 59; and, (iv) a nucleic acid encoding saidprotein, wherein said nucleic acid comprises at least one selector codonthat is recognized by said first O-tRNA; and, (b) incorporating saidunnatural amino acid at said selected position in said protein duringtranslation of said protein in response to said selector codon, therebyproducing said protein comprising said unnatural amino acid at theselected position.
 13. The method of claim 12, wherein said providing atranslation system comprises providing a polynucleotide encoding saidO-RS.
 14. The method of claim 12, wherein said providing a translationsystem comprises providing an O-RS derived from a wild-typeMethanococcus jannaschii tyrosyl-tRNA synthetase.
 15. The method ofclaim 12, wherein said providing a translation system comprises mutatingan amino acid binding pocket of a wild-type aminoacyl-tRNA synthetase bysite-directed mutagenesis, and selecting a resulting O-RS thatpreferentially aminoacylates said O-tRNA with said unnatural amino acid.16. The method of claim 15, wherein said selecting step comprisespositively selecting and negatively selecting for said O-RS from a poolcomprising a plurality of resulting aminoacyl-tRNA synthetase moleculesfollowing site-directed mutagenesis.
 17. The method of claim 12, whereinsaid providing a translation system comprises providing a polynucleotideencoding said O-tRNA.
 18. The method of claim 12, wherein said O-tRNA isan amber suppressor tRNA.
 19. The method of claim 12, wherein saidO-tRNA comprises or is encoded by a polynucleotide sequence set forth inSEQ ID NO: 1 or
 2. 20. The method of claim 12, wherein said selectorcodon is an amber selector codon.
 21. The method of claim 12, furtherwherein said protein comprises a second unnatural amino acid that isdifferent from the first unnatural amino acid, and where providing atranslation system further comprising a second O-RS and a second O-tRNA,wherein the second O-RS preferentially aminoacylates the second O-tRNAwith a second unnatural amino acid that is different from the firstunnatural amino acid, and wherein the second O-tRNA recognizes aselector codon in the nucleic acid that is different from the selectorcodon recognized by the first O-tRNA.
 22. The method of claim 12,wherein said providing a translation system comprises providing a hostcell, wherein said host cell comprises said first unnatural amino acid,said first O-RS, said first O-tRNA and said nucleic acid, and whereinsaid incorporating step comprises culturing said host cell.
 23. Themethod of claim 22, wherein said providing a host cell comprisesproviding a eubacterial host cell or a yeast host cell.
 24. The methodof claim 23, wherein said providing a eubacterial host cell comprisesproviding an E. coli host cell.
 25. The method of claim 22, wherein saidproviding a host cell comprises providing a host cell comprising apolynucleotide encoding said O-RS.
 26. The method of claim 12, whereinsaid providing a translation system comprises providing a cell extract.27. A polypeptide comprising an amino acid sequence at least 90%identical to SEQ ID NO: 59, wherein the polypeptide aminoacylates acognate orthogonal tRNA (O-tRNA) with ap-(2-amino-1-hydroxyethyl)-L-phenylalanine with an efficiency that is atleast 50% of the efficiency observed for a translation system comprisingsaid O-tRNA, said p-(2-amino-1-hydroxyethyl)-L-phenylalanine, and anaminoacyl-tRNA synthetase consisting of the amino acid sequence SEQ IDNO:
 59. 28. A polynucleotide encoding the polypeptide of claim
 27. 29.The polypeptide of claim 27, where said polypeptide is in a cell. 30.The translation system of claim 1, wherein said first O-RS comprises32Asp, 65Glu, 108Arg, 158Gly, and 162Asn.
 31. The translation systemmethod of claim 12, wherein said first O-RS comprises 32Asp, 65Glu,108Arg, 158Gly, and 162Asn.